scholarly journals Disruption of nuclear maturation and rearrangement of cytoskeletal elements in bovine oocytes exposed to heat shock during maturation

Reproduction ◽  
2005 ◽  
Vol 129 (2) ◽  
pp. 235-244 ◽  
Author(s):  
Z Roth ◽  
P J Hansen
Keyword(s):  
Heat Shock ◽  
Subsequent Development ◽  
Meiotic Maturation ◽  
Nuclear Maturation ◽  
Complex Process ◽  
Maturation Medium ◽  
Sphingosine 1 Phosphate ◽  
Series Of Experiments ◽  
Cytoskeletal Elements ◽  
Bovine Oocytes

Meiotic maturation in mammalian oocytes is a complex process which involves extensive rearrangement of microtubules, actin filaments and chromosomes. Since cytoskeletal elements are sensitive to disruption by heat shock, a series of experiments were performed to determine whether physiologically relevant heat shock disrupts the progression of the oocyte through meiosis, fertilization and zygote formation. Cumulus–oocyte complexes were cultured at 38.5, 40.0 or 41.0 °C for the first 12 h of maturation. Incubation during the last 10 h of maturation and 18 h after fertilization was at 38.5 °C and in 5% (v/v) CO2for both treatments. Examination of the cytoskeleton and the chromosome organization in matured oocytes revealed that oocytes matured at 38.5°C were mostly at metaphase II (MII) stage, while the majority of heat-shocked oocytes were blocked at the first metaphase (MI), first anaphase or first telophase stages. A subset of heat-shocked oocytes possessed misshapen MI spindles with disorganized microtubules and unaligned chromosomes. A higher percentage of TUNEL-positive oocytes was noted for oocytes matured at 41.0 °C. Addition of 50 nmol/l sphingosine 1-phosphate to maturation medium blocked the effect of heat shock on progression through meiosis and apoptosis and increased the proportion of oocytes matured at 41.0 °C that were at MII. Following insemination, a high percentage of heat-shocked oocytes were unfertilized, while the majority of the control zygotes were fertilized and had two visible pronuclei. In conclusion, heat shock disrupts nuclear maturation and induces apoptosis. These alterations are likely to be involved in the mechanism underlying heat-shock-induced disruption of oocyte capacity for fertilization and subsequent development.

scholarly journals 298 DISRUPTION OF NUCLEAR MATURATION, APOPTOSIS AND CYTOSKELETAL CHANGES IN BOVINE OOCYTES EXPOSED TO HEAT SHOCK

2005 ◽  
Vol 17 (2) ◽  
pp. 299
Author(s):  
Z. Roth ◽  
P.J. Hansen
Keyword(s):  
Heat Shock ◽  
Temperature Treatment ◽  
Filamentous Actin ◽  
Oocyte Competence ◽  
Nuclear Maturation ◽  
Maturation Medium ◽  
Sphingosine 1 Phosphate ◽  
Series Of Experiments ◽  
Cytoskeletal Elements ◽  
Roche Diagnostics

Heat shock (HS) can cause apoptosis and induce changes in cytoskeletal elements. A series of experiments were performed to determine whether physiologically relevant HS disrupts the progression of oocytes through meiosis, fertilization, and zygote formation, and causes corresponding changes in the cytoskeleton and apoptosis. Cumulus-oocyte complexes (COCs) were cultured at 38.5 (38C), 40 (40C), or 41°C (41C) for the first 12 h of maturation. Incubation during the last 10 h of maturation and 18 h post insemination (hpi) was at 38.5°C and 5% (v/v) CO2 for both treatments. The CATMOD procedure of SAS (SAS Institute, Inc., Cary, NC, USA) was used to analyze the distribution of oocytes into various classes of nuclear maturation and the proportion of apoptotic oocytes. In Exp. 1, matured oocytes were fixed in 4% (w/v) paraformaldehyde, and either stained with Hoechst 33342 or labeled with TUNEL kit (Roche Diagnostics, Indianapolis, IN, USA). Pronuclei were classified as being either condensed or at metaphase I (MI), metaphase II (MII), anaphase I, or telophase I. HS affected (P < 0.001) the distribution of oocytes into stages of meiosis. The majority of 38C oocytes reached MII while 41C oocytes were mostly at MI. Both 40C and 41C increased the percentage of oocytes having TUNEL-positive nuclei (P < 0.001). In Exp. 2, matured oocytes were fixed and stained with Hoechst and markers for either filamentous actin (phalloidin) or microtubules (anti-bovine-α-tubulin labeled with Zenon). Microfilament localization was affected by stage of nuclear maturation and by HS. Actin microfilaments were more prominent in the cytoplasm of heat-shocked oocytes than for 38C oocytes. In addition, the intense ring of actin present under the plasma membrane was reduced for 41C oocytes and the transzonal actin processes present in 38C oocytes were absent in 41C oocytes. A subset of heat-shocked oocytes possessed misshapen MI spindles with disorganized microtubules and unaligned chromosomes. In Exp. 3, addition of 50 nM sphingosine 1-phosphate (S1P) to maturation medium blocked the effect of HS on progression through meiosis and apoptosis. There was a temperature × S1P interaction (P < 0.001) on distribution of oocytes into nuclear classes because S1P increased the proportion of 41C oocytes that were at MII. S1P also blocked the increase in percentage of TUNEL positive oocytes (temperature × treatment, P < 0.005). In Exp. 4, examination of the chromosomal organization for putative zygotes (18 hpi) revealed that HS affected (P < 0.001) their distribution into nuclear classes. The percentage of putative zygotes with a normal diploid pattern was 57% vs. 20% for 38C and 41C oocytes, respectively. In conclusion, HS during the first 12 h of maturation disrupts nuclear maturation, induces apoptosis, alters the cytoskeleteon, and reduces subsequent fertilization. These alterations are likely to be involved in the mechanism underlying heat shock induced disruption of oocyte competence and can be reduced byS1P. This work received the following support: BARD FI-330-2002 and USDA Grant 2002-35203-12664.

scholarly journals Effects of follicle size and electrolytes and glucose in maturation medium on nuclear maturation and developmental competence of bovine oocytes

Reproduction ◽  
2004 ◽  
Vol 127 (2) ◽  
pp. 159-164 ◽  
Author(s):  
Hisataka Iwata ◽  
Shu Hashimoto ◽  
Mayuko Ohota ◽  
Koji Kimura ◽  
Kenichi Shibano ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
Subsequent Development ◽  
Developmental Competence ◽  
Rate Of Development ◽  
Nuclear Maturation ◽  
Maturation Medium ◽  
Follicle Size ◽  
The Difference ◽  
Oviductal Fluid ◽  
Bovine Oocytes

The concentrations of electrolytes (Na, K, Cl, Mg and Ca) and glucose in small follicle (SF) follicular fluid (SFF) and large follicle (LF) follicular fluid (LFF) from slaughterhouse-derived ovaries were studied. Oocytes were matured in medium based on synthetic oviductal fluid. The effects of various concentrations of electrolytes (Na, K, Ca and Mg) and glucose in the maturation medium on the progression of nuclear maturation and subsequent development were also studied. K in SFF was significantly greater than that in LFF. The Mg concentration in follicular fluid (FF) is 2.0–2.3 mM, which is greater than the concentration present in medium generally used for culture. The glucose concentration in FF is about 3.5–3.9 mM and rapidly decreases during the preservation of ovaries. LF oocytes resumed nuclear maturation and progressed to the M2 stage significantly faster than those collected from SF oocytes. In addition, more LF oocytes developed to blastocysts than did SF oocytes. Changing the Na/K ratio in the maturation medium from 16 to 24 did not affect either the progression of nuclear maturation or the rate of development. A low concentration of Mg (0.5 mM) combined with a low Ca concentration (0.5 mM) inhibited the rate of development, but did not affect the progression of nuclear maturation. On the other hand, increasing the Mg concentration to 2.0 mM from 0.5 mM hastened the progression of nuclear maturation and improved the rate of blastulation, irrespective of the Ca concentration. The progression of nuclear maturation was faster and the rate of development was greater with 5.56 mM glucose than with 1.5 mM glucose. The difference in time needed to progress to M2 among the experiment was about 2–3 h. Therefore, prolonging the maturation periods from 21 to 24 h did not change the rate of development. Our results show that the concentrations of Mg and glucose in the maturation medium and the follicle size enveloping the oocyte affect the progression of nuclear maturation and subsequent development. The time requirement for oocytes to reach M2 is strongly related to the developmental competence of the oocytes.

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.

Astaxanthin counteracts the effects of heat shock on the maturation of bovine oocytes

2018 ◽  
Vol 30 (9) ◽  
pp. 1169 ◽  
Author(s):  
J. Ispada ◽  
T. A. Rodrigues ◽  
P. H. B. Risolia ◽  
R. S. Lima ◽  
D. R. Gonçalves ◽  
...  
Keyword(s):  
Heat Shock ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Negative Effects ◽  
Cellular Mechanisms ◽  
Sod Activity ◽  
Oocyte Competence ◽  
Maturation Medium ◽  
Bovine Oocytes

The cellular mechanisms induced by elevated temperature on oocytes are not fully understood. However, there is evidence that some of the deleterious effects of heat shock are mediated by a heat-induced increase in reactive oxygen species (ROS). In this context, carotenoid antioxidants might have a thermoprotective effect. Therefore, the objective of this study was to determine the role of astaxanthin (AST) on oocyte ROS production and on the redox profile and developmental competency of cumulus-oocyte complexes (COCs) after 14 h heat shock (41°C) during in vitro maturation (IVM). Exposure of oocytes to heat shock during IVM increased ROS and reduced the ability of the oocyte to cleave and develop to the blastocyst stage. However, 12.5 and 25 nM astaxanthin rescued these negative effects of heat shock; astaxanthin counteracted the heat shock-induced increase in ROS and restored oocyte developmental competency. There was no effect of astaxanthin on maturation medium lipid peroxidation or on glutathione peroxidase and catalase activity in oocytes and cumulus cells. However, astaxanthin stimulated superoxide dismutase (SOD) activity in heat-shocked cumulus cells. In conclusion, direct heat shock reduced oocyte competence, which was restored by astaxanthin, possibly through regulation of ROS and SOD activity in oocytes and COCs.

scholarly journals Effects of roscovitine on maintenance of the germinal vesicle in horse oocytes, subsequent nuclear maturation, and cleavage rates after intracytoplasmic sperm injection

Reproduction ◽  
2003 ◽  
pp. 693-700 ◽  
Author(s):  
LC Franz ◽  
YH Choi ◽  
EL Squires ◽  
K Hinrichs ◽  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Germinal Vesicle ◽  
Meiotic Maturation ◽  
Developmental Potential ◽  
Nuclear Maturation ◽  
Maturation Medium

This study was conducted to evaluate the effects of roscovitine on suppression of meiosis, subsequent meiotic maturation, and cleavage rates after intracytoplasmic sperm injection of horse oocytes. Oocytes were classified as having compact or expanded cumuli (Com or Exp oocytes) and were divided into three culture groups: 30 h culture in maturation medium (30 h Mat); 54 h culture in maturation medium (54 h Mat), or 24 h culture in medium containing 66 micro mol roscovitine l(-1) and then 30 h culture in maturation medium (Ros+M). After maturation, oocytes were subjected to intracytoplasmic sperm injection and cultured in G1.2 medium for 96 h. Among oocytes fixed immediately after roscovitine culture, 26 of 31 (84%) Com oocytes and 16 of 28 (57%) Exp oocytes were at the germinal vesicle stage (P<0.05). After maturation culture, there were no differences in maturation rates or morphological cleavage rates among treatments. Among Com oocytes, significantly more embryos in the Ros+M treatment than in the 54 h Mat treatment had cleaved with > or = two normal nuclei (63 versus 36%; P<0.05); whereas among Exp oocytes, significantly more embryos in the 30 h Mat treatment than in the Ros+M treatment (63 versus 42%; P<0.05) had cleaved with > or = two normal nuclei. The average number of nuclei in embryos at 96 h was significantly higher (P<0.05) in Ros+M Com oocytes (13.5) than in any other Com or Exp group. These results demonstrate that roscovitine can reversibly maintain equine oocytes in the germinal vesicle stage for up to 24 h, and that such suppression may increase the developmental potential of Com, but not Exp, oocytes.

The effects of cysteine addition during in vitro maturation on the developmental competence, ROS, GSH and apoptosis level of bovine oocytes exposed to heat stress

Zygote ◽  
2011 ◽  
Vol 20 (3) ◽  
pp. 249-259 ◽  
Author(s):  
Hisashi Nabenishi ◽  
Hiroshi Ohta ◽  
Toshihumi Nishimoto ◽  
Tetsuo Morita ◽  
Koji Ashizawa ◽  
...  
Keyword(s):  
Heat Stress ◽  
Formation Rate ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Control Group ◽  
Blastocyst Formation ◽  
Nuclear Maturation ◽  
Maturation Medium ◽  
Bovine Oocytes

SummaryIn the present study, we investigated the effects of various concentrations of cysteine (0.0, 0.6, 1.2 and 1.8 mM) added to the maturation medium on nuclear maturation and subsequent embryonic development of bovine oocytes exposed to heat stress (HS: set at 39.5 °C for 5 h, 40.0 °C for 5 h, 40.5 °C for 6 h, and 40.0 °C for 4 h versus 38.5 °C for 20 h as the control group). This regime mimicked the circadian rhythm of the vaginal temperature of lactating dairy cows during the summer season in southwestern Japan. Moreover, we also evaluated the oocyte's reactive oxygen species (ROS) and glutathione (GSH) levels and the apoptosis levels of the oocytes and cumulus cells in the presence or absence of 1.2 mM cysteine. As a result, HS in the without-cysteine group significantly suppressed (p < 0.05) both the nuclear maturation rate up to the metaphase (M)II stage and the blastocyst formation rate compared with that of the control group. In addition, this group showed significantly higher (p < 0.05) ROS levels and significantly lower (p < 0.05) GSH levels than those of the control group. Moreover, the level of TdT-mediated dUTP nick end labelling (TUNEL)-positive cumulus cells in the HS without-cysteine group was significantly higher (p < 0.05) than that of the control group. However, the addition of 1.2 mM cysteine to the maturation medium restored not only the nuclear maturation, blastocyst formation rates and GSH contents, but also increased the ROS and TUNEL-positive levels of the cumulus cells, but not oocytes, to that of the control group. These results indicate that the addition of 1.2 mM cysteine during in vitro maturation (IVM) may alleviate the influence of heat stress for oocyte developmental competence by increasing GSH content and inhibiting the production of oocyte ROS followed by apoptosis of cumulus cells.

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

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