Differential effects of linoleic and alpha-linolenic fatty acids on spatial and temporal mitochondrial distribution and activity in bovine oocytes

2012 ◽  
Vol 24 (5) ◽  
pp. 679 ◽  
Author(s):  
Waleed F. Marei ◽  
D. Claire Wathes ◽  
Ali A. Fouladi-Nashta
Keyword(s):  
Membrane Potential ◽  
Oocyte Maturation ◽  
Inhibitory Effect ◽  
Inner Membrane ◽  
Nuclear Maturation ◽  
Mitochondrial Inner Membrane ◽  
Maturation Rate ◽  
Microscope Imaging ◽  
Mitochondrial Distribution ◽  
Bovine Oocytes

Using specific stains and confocal microscope imaging, the patterns of mitochondrial distribution, mitochondrial inner membrane potential and reactive oxygen species (ROS) levels during bovine oocyte maturation were determined in the presence or absence of physiological concentrations of linoleic acid (LA; 100 µM) or α-linolenic acid (ALA; 50 µM). Mitochondrial distribution in control oocytes at 0 h was mainly peripheral and changed to a diffused pattern after 1 h of culture; this was maintained up to 24 h. Mitochondrial clusters were observed during the early hours of maturation (1–4 h); the majority of these were arranged in perinuclear fashion. LA supplementation resulted in: (1) delayed redistribution of the mitochondria from a peripheral to a diffuse pattern and a decreased percentages of oocytes showing perinuclear mitochondrial clusters, (2) decreased mitochondrial inner membrane potential at 1 and 24 h compared with the control and (3) higher ROS levels, associated with a lower nuclear maturation rate. In contrast, ALA supplementation had no effect on mitochondrial distribution and activity and decreased ROS levels compared with the control; this was associated with an increased nuclear maturation rate. In conclusion, LA induced alterations in mitochondrial distribution and activity as well as increasing ROS levels, which mediate, at least in part, the inhibitory effect on oocyte maturation.

167 Effect of Storage Time and Temperature of a Commercial Embryo Holding Medium on the Maturation Kinetics of Immature Bovine Oocytes

2018 ◽  
Vol 30 (1) ◽  
pp. 223
Author(s):  
O. B. Pascottini ◽  
M. Catteeuw ◽  
A. Van Soom ◽  
G. Opsomer
Keyword(s):  
Oocyte Maturation ◽  
Germinal Vesicle ◽  
Holding Time ◽  
Control Group ◽  
Nuclear Maturation ◽  
Maturation Medium ◽  
Maturation Rate ◽  
Maturation Stages ◽  
Bovine Oocytes ◽  
Kinetics Of

The effect of holding time and temperature during storage of immature bovine oocytes in a commercial embryo holding medium (EHM; Syngro® Ltd., Livingston, United Kingdom) was evaluated. Ovaries were collected at the local slaughterhouse and processed within 2 h. Cumulus-oocyte complexes (COC) were collected and allocated to groups of 60. The COC were held in 1-mL sterile glass osmometer tubes, filled to the top with the EHM to limit the amount of air. Vials were capped and covered with parafilm to ensure a tight seal and prevent leakage. Tubes were stored for 6 h at 4°C, room temperature (RT), or 38.5°C; for 10 h at 4°C and RT; and for 14 h at RT. Next, oocytes were fixed after storage in EHM (immature holding) or fixed after being held in EHM and subsequent 22-h maturation at 38.5°C in 5% CO2 in humidified air (mature holding). Maturation medium consisted of modified bicarbonate-buffered TCM-199 supplemented with gentamycin and epidermal growth factor. During all experiments, a control group was included each time. The control consisted of groups of 60 COC immediately fixed after collection or transferred to maturation medium for 22 h and subsequently fixed. Nuclear maturation of oocytes was assessed after Hoechst 33342 staining, using a 400× magnification fluorescence microscope. A total of 3043 COC were evaluated in 3 replicates. Oocytes maturation stages were classified as (1) oocytes in germinal vesicle stage, (2) oocytes in meiotic progression (diakinesis, metaphase I, or anaphase), (3) matured (telophase I or metaphase II), and (4) degenerated (degraded chromatin). Oocytes remained at the germinal vesicle stage when held in EHM (without subsequent maturation) regardless of holding time and temperature (P > 0.05). When oocytes were held for 6 h and subsequently matured (Table 1), the number of matured oocytes was significantly lower for oocytes held at 38.5°C compared with the other groups (control, RT, and 4°C). When held for 10 h, the oocyte maturation rate was similar between the control and RT groups (P > 0.05), but it was significantly lower in oocytes held at 4°C. Last, when compared with oocytes held at RT for 14 h, the maturation rate was higher in the control group (P < 0.05). To conclude, immature bovine oocytes can be successfully held in EHM at RT for up to 10 h. Storing immature oocytes in EHM can delay oocyte maturation and concomitantly synchronize maturation. Table 1.Kinetics of cumulus-oocyte complex nuclear status after storage in embryo holding medium for different times and temperatures and subsequent 22-h maturation

Abstract 332: Mitochondrial Inner-Membrane Potential Instability Promotes Sarcolemmal Electrical Instability after Ischemia-Reperfusion in Monolayers of Cardiac Myocytes

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Soroosh Solhjoo ◽  
Brian O’Rourke
Keyword(s):  
Membrane Potential ◽  
Cardiac Myocytes ◽  
Mitochondrial Function ◽  
Ischemia Reperfusion ◽  
Neonatal Rat ◽  
Partial Recovery ◽  
Mitochondrial Network ◽  
Electrical Excitability ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane

Mitochondrial uncoupling due to oxidative stress can, through opening of sarcolemmal KATP channels, alter cellular electrical excitability, and it has been proposed that mitochondrial function is a major factor in arrhythmogenesis during ischemia-reperfusion. Here, we examine the effects of ischemia-reperfusion on mitochondrial inner membrane potential (ΔΨm) and corresponding changes in electrical excitability and wave propagation in monolayer cultures of neonatal rat ventricular myocytes. Changes in ΔΨm were observed using TMRM and changes in the sarcolemmal voltage were recorded with a 464-element photodiode array using di-4-ANEPPS. Ischemia was induced by covering the center part of the monolayer (D = 22 mm) with a coverslip (D = 15 mm). Cell contractions ceased after approximately 6 min of ischemia; however, electrical activity continued for 11.3 ± 4.2 min (N = 5). Amplitude and conduction velocity of the action potentials in the ischemic region decreased over the same time period. ΔΨm was lost in two phases: a reversible phase of partial depolarization, after 11.2 ± 1.3 min of ischemia, and a nonreversible phase, which happened after 30 ± 6 min of ischemia, during which the whole mitochondrial network of the myocyte became depolarized and the cells underwent contracture (N = 4). Reperfusion after the long ischemia resulted in only partial recovery and the observance of oscillations of ΔΨm in the mitochondrial network or rapid flickering of individual mitochondrial clusters and was associated with heterogeneous electrical recovery, and formation of wavelets and reentry (4/5 monolayers). In contrast, mitochondria fully recovered and reentry was rare (1/5 monolayers) for reperfusion after the short ischemia (10-12 min). 4’-chlorodiazepam, an inhibitor of inner membrane anion channels, stabilized mitochondrial function after the long ischemia, and prevented wavelets (5/5 monolayers) and reentry (4/5 monolayers). In conclusion, incomplete or unstable recovery of mitochondrial function after ischemia correlates with reentrant arrhythmias in monolayers of cardiac myocytes. Our findings suggest that stabilization of mitochondrial network dynamics is an important strategy for preventing ischemia/reperfusion-related arrhythmias.

scholarly journals Effect of Hyaluronan on Developmental Competence and Quality of Oocytes and Obtained Blastocysts fromIn VitroMaturation of Bovine Oocytes

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Jolanta Opiela ◽  
Joanna Romanek ◽  
Daniel Lipiński ◽  
Zdzisław Smorąg
Keyword(s):  
Dna Fragmentation ◽  
Oocyte Maturation ◽  
Developmental Competence ◽  
Meiotic Maturation ◽  
Nuclear Maturation ◽  
Significant Difference ◽  
The Mean ◽  
Bovine Oocytes

The objective of the present study was to evaluate the effect of hyaluronan (HA) during IVM on meiotic maturation, embryonic development, and the quality of oocytes, granulosa cells (GC), and obtained blastocysts. COCs were maturedin vitroin control medium and medium with additional 0.035% or 0.07% of exogenous HA. The meiotic maturity did not differ between the analysed groups. The best rate and the highest quality of obtained blastocysts were observed when 0.07% HA was used. A highly significant difference (P<0.001) was noted in the mean number of apoptotic nuclei per blastocyst and in the DCI between the 0.07% HA and the control blastocysts (P<0.01). Our results suggest that addition of 0.035% HA and 0.07% HA to oocyte maturation media does not affect oocyte nuclear maturation and DNA fragmentation. However, the addition of 0.07% HA during IVM decreases the level of blastocysts DNA fragmentation. Finally, our results suggest that it may be risky to increase the HA concentration during IVM above 0.07% as we found significantly higherBaxmRNA expression levels in GC cultured with 0.07% HA. The final concentration of HA being supplemented to oocyte maturation media is critical for the success of the IVP procedure.

174 Effects of Melatonin on In Vitro Maturation of Bovine Oocytes and Gene Expression in Cumulus Cells: Preliminary Results

2018 ◽  
Vol 30 (1) ◽  
pp. 226
Author(s):  
F. C. Castro ◽  
L. Schefer ◽  
K. L. Schwarz ◽  
H. Fernandes ◽  
R. C. Botigelli ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Vitro Culture ◽  
Reverse Transcription ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Nuclear Maturation ◽  
Maturation Rate ◽  
Bovine Oocytes

Melatonin mediates several processes in animal reproduction and has drawn attention for its potent antioxidant, anti-apoptotic, anti-inflammatory action and, more recently, for its benefits on oocyte maturation and embryo development in vitro. The aim of this study was to assess the effect of melatonin during the in vitro maturation (IVM) on nuclear maturation of bovine oocytes and gene expression in their corresponding cumulus cells (CC). Bovine cumulus–oocyte complexes (COC) were obtained by aspiration of follicles (2-6 mm) from slaughterhouse ovaries, selected (grades I and II) and transferred to 4 well plates (25-30 COC/well) containing IVM medium [TCM-199 supplemented with sodium bicarbonate (26 mM), sodium pyruvate (0.25 mM), FSH (0.5 µg mL−1), LH (5.0 µg mL−1), 0.3% BSA, and gentamicin (50 µg mL−1)] with 0, 10−5, 10−7, 10−9 or 10−11 M melatonin and cultured for 24 h at 38.5°C and 5% CO2. At the end of IVM, oocytes were stained with Hoechst 33342 (10 μg mL−1) and evaluated for nuclear maturation rate. The CC were evaluated for the expression of antioxidant (SOD1, SOD2, GPX4), pro-apoptotic (P53, BAX) and expansion-related genes (PTX3, HAS1, HAS2). For transcript detection in CC, RNA isolation was performed with TRIzol®Reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcription with High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). Relative quantification of transcripts was performed by RT-qPCR using 3 endogenous controls (β-actin, GAPDH, PPIA). Nuclear maturation rate and gene expression were tested by ANOVA and means were compared by Tukey’s test (6 replicates). In CC, the different concentrations of melatonin did not significantly alter expression of the investigated genes (P > 0.05), although all concentrations provided a numerical increase in the expression of the antioxidant SOD1 and of the expansion-related genes PTX3 and HAS2. Regarding the pro-apoptotic genes, concentrations of 10−11 and 10−9 M were able to reduce only numerically the expression of BAX and P53, respectively. In oocytes, the rate of nuclear maturation was not different among the tested treatments (P > 0.05), but it was numerically higher in the 10−7 M melatonin treated group compared with the control (69.71 ± 13.76% v. 88.1 ± 12.54%). In conclusion, under the studied conditions, melatonin was unable to improve maturation rate or to affect the expression of antioxidant, pro-apoptotic, and expansion-related genes in CC. Melatonin during IVM has shown variable results in different studies and appears to show different effects depending on culture conditions and parameters studied. In order to take advantage of the possible positive antioxidant effects of melatonin, other culture conditions and parameters should be investigated. In a next step, melatonin will be included during in vitro culture of embryos to evaluate its possible cytoprotective role, because such embryos are more exposed to oxidative stress during in vitro culture, and to investigate its benefits on developmental competence in vitro. This reaesrch was funded by FAPESP (2015/20379-0; 2014/17181-0).

scholarly journals The translocator maintenance protein Tam41 is required for mitochondrial cardiolipin biosynthesis

2008 ◽  
Vol 183 (7) ◽  
pp. 1213-1221 ◽  
Author(s):  
Stephan Kutik ◽  
Michael Rissler ◽  
Xue Li Guan ◽  
Bernard Guiard ◽  
Guanghou Shui ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Respiratory Chain ◽  
Matrix Protein ◽  
Mitochondrial Protein ◽  
Pleiotropic Effects ◽  
Mitochondrial Matrix ◽  
Carrier Proteins ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane

The mitochondrial inner membrane contains different translocator systems for the import of presequence-carrying proteins and carrier proteins. The translocator assembly and maintenance protein 41 (Tam41/mitochondrial matrix protein 37) was identified as a new member of the mitochondrial protein translocator systems by its role in maintaining the integrity and activity of the presequence translocase of the inner membrane (TIM23 complex). Here we demonstrate that the assembly of proteins imported by the carrier translocase, TIM22 complex, is even more strongly affected by the lack of Tam41. Moreover, respiratory chain supercomplexes and the inner membrane potential are impaired by lack of Tam41. The phenotype of Tam41-deficient mitochondria thus resembles that of mitochondria lacking cardiolipin. Indeed, we found that Tam41 is required for the biosynthesis of the dimeric phospholipid cardiolipin. The pleiotropic effects of the translocator maintenance protein on preprotein import and respiratory chain can be attributed to its role in biosynthesis of mitochondrial cardiolipin.

169 Nuclear Maturation Kinetics and In Vitro Fecundation of Immature Bovine Oocytes Injected into Preovulatory Follicles

2018 ◽  
Vol 30 (1) ◽  
pp. 224
Author(s):  
L. M. S. Simoes ◽  
A. P. C. Santos ◽  
E. A. Lima ◽  
R. E. Orlandi ◽  
M. P. Bottino ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Germinal Vesicle ◽  
Causative Factor ◽  
Nuclear Maturation ◽  
Preovulatory Follicles ◽  
Fertilization Capacity ◽  
Bovine Oocytes ◽  
Metaphase I

The objective was to evaluate in vitro nuclear maturation and fecundation kinetics of oocytes injected into preovulatory follicles of synchronized cows using the intra-follicular oocyte injection (IFOI) technique. In experiment 1, 438 immature abattoir-bovine cumulus–oocyte complexes (COC) of grades I, II, and III were randomly allocated to 1 of 3 groups: Matvitro (n = 111), COC matured in vitro for 22 h; Matvivo20 (n = 172) and Matvivo30 (n = 155), 30 oocytes were injected into each preovulatory follicle of pre-synchronized recipients. In Matvivo20, oocytes were matured for 19.8 ± 0.1 h and in Matvivo30, for 28.3 ± 0.1 h. All cows received 12.5 mg of LH (Lutropin, Bioniche, Canada) at IFOI (Matvivo20) or 10 h after IFOI (Matvivo30). Oocytes from Matvivo20 and Matvivo30 were aspirated 20 h after LH injection for assessment of oocyte maturation and recovery rates. Oocytes were evaluated according to maturation kinetics as germinal vesicle, metaphase I, anaphase I, telophase I, metaphase II, parthenogenetically activated, and degenerated (chromosomal aberrations, presence of diffuse or indefinite chromatin). In experiment 2, immature abattoir-bovine COC (n = 202) of grades I, II, and III were randomly distributed into 2 groups: Matvitro (n = 103), COC were matured and fertilized in vitro; Matvivo (n = 99), same as Matvivo20 protocol, and COC fertilized in vitro. Presumptive zygotes were evaluated as fertilized, unfertilized, or polyspermic. Statistical analyses were performed by the GLIMMIX procedure of SAS (SAS Institute Inc., Cary, NC, USA). Recovery rate was lower (P < 0.001) in Matvivo20 (52.9%, 91/172) compared with Matvivo30 (72.9%, 113/155). Germinal vesicle (P = 0.94), metaphase I (P = 0.98), anaphase I (P = 0.99), and telophase I (P = 0.20) rates were similar. However, there were differences in metaphase II [Matvitro: 81.0% (90/111)a, Matvivo20: 74.5% (35/47)a, and Matvivo30: 41.6% (32/77)b; P = 0.001], degenerate [Matvitro: 5.4% (6/111)c, Matvivo20: 21.3% (10/47)b and Matvivo30: 48.1% (37/77); P = 0.001] and parthenogenetically activated [Matvitro: 0.0% (0/111)b, Matvivo20: 0.0% (0/47)b and Matvivo30: 9.1% (7/77)a; P = 0.001]. Polyspermic (P = 0.18) and abnormal (P = 0.98) rates were similar. However, there was a higher rate (P = 0.05) of fertilized oocytes in Matvivo (60.6%, 60/99) than in Matvitro (46.6%, 48/103). In conclusion, oocyte maturation in vivo after IFOI for 20 h does not alter maturation kinetics and increases in vitro oocyte fertilization capacity. However, the 10-h increase in intra-follicular oocyte permanence decreased the proportion of viable oocytes. Thus, the oocyte maturation phase is not the limiting causative factor for the low IFOI-embryo production rates.

Effects of ghrelin on activation of Akt1 and ERK1/2 pathways during in vitro maturation of bovine oocytes

Zygote ◽  
2017 ◽  
Vol 25 (2) ◽  
pp. 183-189 ◽  
Author(s):  
Thomas-Markos Chouzouris ◽  
Eleni Dovolou ◽  
Fotini Krania ◽  
Ioannis S. Pappas ◽  
Konstantinos Dafopoulos ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Cumulus Cells ◽  
Treated Group ◽  
Control Group ◽  
Dependent Manner ◽  
Nuclear Maturation ◽  
Bovine Oocytes ◽  
Phosphorylation Rate ◽  
Matured Oocyte

SummaryThe purpose of this study was to investigate the possible molecular pathways through which ghrelin accelerates in vitro oocyte maturation. Bovine cumulus–oocyte complexes (COCs), after 18 or 24 h maturation in the absence or the presence of 800 pg ml–1 of acylated ghrelin were either assessed for nuclear maturation or underwent in vitro fertilization in standard media and putative zygotes were cultured in vitro for 8 days. In a subset of COCs the levels of phosphorylated Akt1 and ERK1/2 (MAPK1/3) were assessed at the 0th, 6th, 10th, 18th and 24th hours of in vitro maturation (IVM). At 18 and 24 h no difference existed in the proportion of matured oocytes in the ghrelin-treated group, while in the control group more (P < 0.05) matured oocyte were found at 24 h. Oocyte maturation for 24 h in the presence of ghrelin resulted in substantially reduced (P < 0.05) blastocyst yield(16.3%) in comparison with that obtained after 18 h (30.0%) or to both control groups (29.3% and 26.9%, for 18 and 24 h in maturation, respectively). Ghrelin-treated oocytes expressed lower Akt1 phosphorylation rate at the 10th hour of IVM, and higher ERK1/2 at the 6th and 10th hours of IVM compared with controls. In cumulus cells, at the 18th and 24th hours of IVM Akt1 phosphorylation rate was higher in ghrelin-treated oocytes. Our results imply that ghrelin acts in a different time-dependent manner on bovine oocytes and cumulus cells modulating Akt1 and ERK1/2 phosphorylation, which brings about acceleration of the oocyte maturation process.

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