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 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.

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.

85 The effects of heat exposure on the growth and developmental competence of oocytes derived from early antral follicles in dairy cows

2021 ◽  
Vol 33 (2) ◽  
pp. 150
Author(s):  
K. Kawano ◽  
K. Sakaguchi ◽  
E. Furukawa ◽  
M. Chelenga ◽  
Y. Yanagawa ◽  
...  
Keyword(s):  
Heat Stress ◽  
Heat Shock ◽  
Dairy Cows ◽  
Shock Group ◽  
Developmental Competence ◽  
Control Group ◽  
Oocyte Competence ◽  
Antral Follicles ◽  
Nuclear Maturation ◽  
Summer Heat

Summer heat stress in dairy cows impairs the developmental competence of oocytes from antral follicles (2–8mm) which are used in conventional IVM and IVF systems. Moreover, summer heat stress is considered to impair the oocyte competence derived from smaller follicles; therefore, the impairment of oocyte competence possibly continues into the cooler autumn season. To investigate the thermosensitivity of early antral follicles (&lt;1mm), we evaluated the effects of heat exposure on the growth and developmental competence of oocytes using invitro culture of oocyte–cumulus-granulosa complexes (OCGCs) derived from early antral follicles. OCGCs (n=315) were collected from early antral follicles (0.5–1mm) and cultured for 12 days. OCGCs in the heat shock group were cultured using a temperature cycle of 38.5°C for 5h, 39.5°C for 5h, 40.5°C for 5h, and 39.5°C for 9h, whereas those in the control group were cultured at a constant temperature of 38.5°C for 24h. The diameters of oocytes were measured before culture. Half of the culture medium was replaced every 4 days. Oestradiol-17β (E2) and progesterone (P4) production during the first, second, and third 4-day periods were measured by enzyme immunoassay; the viability of OCGCs was evaluated based on their morphology. Oocytes that survived after 12 days of culture (n=191) were subjected to IVM (38.5°C, 22h); their diameter and nuclear status were evaluated. Some oocytes (n=71) were subjected to IVF (38.5°C, 18h) and embryo culture (39.0°C, 150h). Cleavage and blastocyst rates were evaluated at 48h and 168h after IVF. Effects of treatment groups and culture periods on E2 and P4 production and diameters of oocytes were evaluated by two-way ANOVA followed by Tukey-Kramer or Student’s t-test. The viability of OCGCs, nuclear maturation, cleavage and blastocyst rates between two groups were compared by the chi-squared or Fisher’s exact test. E2 and P4 production and the viability of OCGCs were not different between the 2 groups. Although mean oocyte diameters before culture did not differ between the 2 groups, the mean diameters after IVM were significantly smaller in the heat shock group (108.0µm, n=56) than in the control group (111.7µm, n=61; P&lt;0.05). The nuclear maturation rate in the heat shock group (36.4%, n=55) was significantly lower than in the control group (60.3%, n=58; P&lt;0.05). Cleavage rates were similar between the control (54.5%, n=33) and heat shock groups (45.7%, n=35). However, no oocytes developed to blastocysts in the heat shock group (0%, n=35), whereas 30.3% (n=33) oocytes developed to blastocysts (cell number±s.d.; 92.4±28.4) in the control group (P&lt;0.05). These findings suggest that summer heat stress in dairy cows impairs the growth, nuclear maturation, and developmental competence of oocytes derived from early antral follicles. This experimental model could be used to explore the mechanisms by which heat stress subsequently impairs oocyte competence during the cooler autumn season.

162 Supplementation of Fibroblast Growth Factor 2, Leukemia Inhibitory Factor, and Insulin-Like Growth Factor 1 (FLI) Enables Efficient Production of Competent Porcine Oocytes Without Gonadotropins in the Maturation Medium

2018 ◽  
Vol 30 (1) ◽  
pp. 220
Author(s):  
Y. Yuan ◽  
L. D. Spate ◽  
B. K. Redel ◽  
R. S. Prather ◽  
R. M. Roberts
Keyword(s):  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Leukemia Inhibitory Factor ◽  
Fibroblast Growth Factor 2 ◽  
Fibroblast Growth ◽  
Blastocyst Formation ◽  
Oocyte Competence ◽  
Nuclear Maturation ◽  
Maturation Medium

In vitro maturation (IVM) of oocytes is a critical step in assisted reproductive technologies carried out in species such as cattle and swine, for generating oocytes capable of being fertilized in vitro and providing healthy offspring useful for biomedical and agricultural purposes. Cumulus-oocyte complexes (COC) collected from abattoir ovaries for IVM often respond poorly to gonadotropins, resulting in compromised oocyte competence. Our previous work demonstrated that the combination of fibroblast growth factor 2, leukemia inhibitory factor, and insulin-like growth factor 1 (FGF2, LIF, and IGF1; FLI) significantly improved nuclear maturation of porcine oocytes and their developmental competence. However, it is unclear whether the benefits of FLI are mediated through increased gonadotropin sensitivity of COC or improved downstream signalling. Here we investigated the effect of FLI supplementation of IVM medium with and without gonadotropins. The COC, collected from 2- to 6-mm follicles from prepubertal ovaries, were matured in 5% CO2/air at 38.5°C for 42 h in chemically defined TCM-199 medium (supplemented with 0.1% polyvinyl alcohol, 3.05 mM d-glucose, 0.91 mM pyruvate, 0.57 mM cysteine, 10 ng mL−1 epidermal growth factor), with or without FLI (40 ng mL−1 FGF2, 20 ng mL−1 LIF, and 20 ng mL−1 IGF1), and with or without gonadotropins (0.5 μg mL−1 LH, 0.5 μg mL−1 FSH), in a 2 × 2 factorial design experiment. After IVM, oocytes were fertilized in vitro and cultured under standard conditions until Day 6 when blastocyst formation was assessed. The experiment was replicated 4 times with a total of 792 oocytes. Percentage data were arcsin transformed and analysed by ANOVA to detect differences (significance, P < 0.05). When FLI was absent from the maturation medium, oocytes matured in presence of gonadotropins demonstrated improved nuclear maturation (67.0 ± 2.2% v. 52.7 ± 5.3%) and blastocyst formation (23.7 ± 3.3% v. 10.2 ± 2.3%) relative to oocytes matured without gonadotropins, respectively. However, when FLI was present in the medium, the extent of oocyte maturation and subsequent blastocyst development was unaffected by gonadotrophin addition (maturation: 84.2 ± 1.8% with and 79.2 ± 3.4% without; blastocyst formation: 29.5 ± 4.3% with and 24.9 ± 4.3% without). In summary, these results suggest that FLI, rather than enhancing COC sensitivity to gonadotrophins, may activate certain downstream signalling pathways that are normally controlled by gonadotropins during IVM, thereby enhancing oocyte quality. Therefore, FLI appears able to substitute for gonadotropins in the maturation medium and supports porcine oocyte competence in the absence of FSH and LH. These observations will help us better understand the mechanisms whereby FLI enhances oocyte nuclear maturation and improves developmental competency. Supported by NIH R01HD69979, U42OD011140, and University of Missouri Food for the 21st Century Program.

266 Effects of Inflammatory States on Ovarian Biology and Oocyte Competence

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 134-135
Author(s):  
Jennifer A Hernandez Gifford ◽  
Emily Ferranti ◽  
Kylee Forrest ◽  
Craig A Gifford
Keyword(s):  
Granulosa Cell ◽  
Oocyte Maturation ◽  
Beta Catenin ◽  
Control Mechanisms ◽  
Steroid Production ◽  
Oocyte Competence ◽  
Nuclear Maturation ◽  
Naturally Occurring ◽  
Hormonal Milieu

Abstract Female fertility is dependent on estradiol concentrations which regulate a multitude of ovarian functions including follicle development and oocyte maturation leading to ovulation of a viable oocyte. Estradiol biosynthesis is regulated by coordinated actions of follicle-stimulating hormone and intra-ovarian control mechanisms including the co-transcription factor beta-catenin. Beta-catenin is a multi-faceted protein recognized for its role in granulosa cell steroid production and is shown to be modulated by lipopolysaccharide (LPS), the endotoxin responsible for stimulation of the immune system in infections caused by Gram-negative bacteria. Beef heifers treated with subacute concentrations of LPS during a synchronized follicular wave demonstrate a decline in serum estradiol concentrations 50 h after CIDR withdrawal, corresponding with dominant follicle maturation and preceding ovulation. The endotoxin exposure also resulted in increased LPS concentration and E2:P4 ratios in follicular fluid suggesting that low dose LPS modulates the intra-follicular hormonal milieu. Additionally, in a granulosa cell line, LPS treatment decreased mRNA expression of aromatase and beta-catenin. These data indicate that LPS alters E2 synthesis by modulating beta-catenin and subsequent steroidogenic enzyme expression. To further explore the contribution of naturally occurring LPS exposure on follicular steroid production and developing oocytes, bovine ovary pairs were collected from local abattoirs. Oocytes were aspirated from small follicles and matured in vitro to evaluate meiotic events related to nuclear maturation and spindle morphology. Small follicles from ovarian pairs were separated by the detectable LPS concentrations into high and low LPS groups. Oocytes matured from low LPS follicles demonstrated an increase in the percent of abnormal maturation events. Data indicate that induced or naturally occurring low doses of LPS can alter circulating and follicular estradiol concentrations impairing oocyte maturation. Perturbation to local ovarian signaling cascades from subclinical inflammatory disease may be an unappreciated factor altering fertility and leading to decreased cow retention.

Effects of oviductal proteins, including heat shock 70 kDa protein 8, on survival of ram spermatozoa over 48 h in vitro

2009 ◽  
Vol 21 (3) ◽  
pp. 408 ◽  
Author(s):  
R. E. Lloyd ◽  
R. M. A. Elliott ◽  
A. Fazeli ◽  
P. F. Watson ◽  
W. V. Holt
Keyword(s):  
Plasma Membrane ◽  
Heat Shock ◽  
Membrane Proteins ◽  
Epithelial Cells ◽  
Beneficial Effect ◽  
Active Component ◽  
Apical Plasma Membrane ◽  
Series Of Experiments ◽  
Dose Dependent

Following insemination, ram spermatozoa are transported to the isthmus region of the oviduct where they bind to the oviductal epithelial cells (OEC), remaining viable for several hours. The aim of the present study was to begin to decipher which component(s) of the ewe oviduct actively participates in maintaining the viability of ram spermatozoa. A series of experiments was conducted to investigate whether: (1) soluble OEC apical plasma membrane proteins (sAPM) isolated from ewes prolong survival of ram spermatozoa over an extended (48 h) coincubation period at 39°C; (2) a recombinant form of one of these oviductal proteins, namely heat shock 70 kDa protein 8 (HSPA8), prolongs survival of ram spermatozoa; and (3) pretreatment with HSPA8 antibody compromises the ability of sAPM to prolong the survival of ram spermatozoa. Both sAPM and recombinant HSPA8 had a beneficial effect on the viability of ram spermatozoa during coincubation, although both these effects were dose dependent. In contrast, pretreatment with HSPA8 antibody significantly negated the ability of sAPM to maintain the viability of ram spermatozoa. These findings suggest that HSPA8 is an active component of the ewe oviduct that participates in maintaining the viability of ram spermatozoa. This is a potentially valuable observation given that there is a great deal of room for improving existing diluents for storing fresh ram semen.

scholarly journals 324SPHINGOSINE-1-PHOSPHATE PROTECTS CULTURED BOVINE OOCYTES FROM PHYSIOLOGICALLY RELEVANT THERMAL STRESS

2004 ◽  
Vol 16 (2) ◽  
pp. 282 ◽  
Author(s):  
Z. Roth ◽  
P.J. Hansen
Keyword(s):  
Thermal Stress ◽  
Heat Shock ◽  
Blastocyst Stage ◽  
Caspase Activity ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
Oocyte Competence ◽  
Cell Death Pathway

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that can block the sphingomyelin cell-death pathway by suppressing ceramide-induced apoptosis. The present study was performed to test whether S1P protects oocytes from heat shock during in vitro maturation. Cumulus-oocyte complexes obtained by slicing follicles were placed in maturation medium with or without 50nM S1P and cultured at 38.5°C (CON) or 41°C (41C) for the first 12h of maturation. Incubation during the last 10h of maturation (22-h total maturation time), fertilization, and embryonic development were performed at 38.5°C and 5% (v/v) CO2. Blastocyst development was recorded at 8 days post-insemination (dpi) and activity of group II caspases in 8-day blastocysts was determined using a fluoroprobe, PhiPhiLux-G1D2 (OncoImmunin, Gaithersburg, MD, USA). Data were analysed by least-squares ANOVA with the GLM procedure of SAS. Percentage data were subjected to arcsin transformation before analysis. Exposure of oocytes to thermal stress during the first 12h of maturation reduced cleavage rate (P&lt;0.01) and the number of oocytes developing to the blastocyst stage (P&lt;0.04). There was a temperature x S1P interaction for cleavage rate (P&lt;0.03) because S1P blocked effects of thermal stress on cleavage rate. Without S1P, the percentage of oocytes that cleaved by 3 dpi were 83.6±2.7% and 65.8±2.7% for CON and 41C, respectively. In the presence of S1P, percent cleavage was 86.7±2.7% and 83.9±2.7% for CON and 41C, respectively. There was a trend (P=0.06) for a temperature x S1P interaction for percent oocytes developing to blastocyst stage because S1P blocked effects of heat shock on development. Without S1P, the percentages of oocytes that developed to the blastocyst stage were 28.7±3.0% and 15.2±3.0% for CON and 41C, respectively. In the presence of S1P, percent blastocysts were 24.3±3.4% and 23.9±3.0% for CON and 41C, respectively. When development was expressed as percentage of cleaved embryos, however, there were no effects of temperature, S1P, or temperature x S1P on percent development to the blastocyst stage. Blastocyst caspase activity was not affected by temperature or S1P. In summary, exposure to physiologically relevant thermal stress during the first 12h of maturation has a deleterious effect on oocyte competence and this effect can be reduced by S1P. The fact that heat shock reduced the percentage of oocytes but not the percentage of cleaved embryos that became blastocysts suggests that oocytes that survive effects of heat shock and cleave have normal potential to develop to the blastocyst stage. Moreover, since heat shock did not affect caspase activity, it is likely that blastocysts from heat-shocked oocytes have normal developmental potential, at least as determined by caspase activity. Support: BARD FI-330-2002 and USDA Grants 2002-35203-12664 and 2001-52101-11318.

scholarly journals Oocyte Competence Biomarkers Associated With Oocyte Maturation: A Review

2021 ◽  
Vol 9 ◽  
Author(s):  
Batara Sirait ◽  
Budi Wiweko ◽  
Ahmad Aulia Jusuf ◽  
Dein Iftitah ◽  
R. Muharam
Keyword(s):  
Oocyte Maturation ◽  
Blastocyst Stage ◽  
Current Approach ◽  
Developmental Competence ◽  
Matrix Remodeling ◽  
Oocyte Competence ◽  
Nuclear Maturation ◽  
Oocyte Developmental Competence ◽  
Cumulus Oocyte Complex

Oocyte developmental competence is one of the determining factors that influence the outcomes of an IVF cycle regarding the ability of a female gamete to reach maturation, be fertilized, and uphold an embryonic development up until the blastocyst stage. The current approach of assessing the competency of an oocyte is confined to an ambiguous and subjective oocyte morphological evaluation. Over the years, a myriad of biomarkers in the cumulus-oocyte-complex has been identified that could potentially function as molecular predictors for IVF program prognosis. This review aims to describe the predictive significance of several cumulus-oocyte complex (COC) biomarkers in evaluating oocyte developmental competence. A total of eight acclaimed cumulus biomarkers are examined in the study. RT-PCR and microarray analysis were extensively used to assess the significance of these biomarkers in foreseeing oocyte developmental competence. Notably, these biomarkers regulate vital processes associated with oocyte maturation and were found to be differentially expressed in COC encapsulating oocytes of different maturity. The biomarkers were reviewed according to the respective oocyte maturation events namely: nuclear maturation, apoptosis, and extracellular matrix remodeling, and steroid metabolism. Although substantial in vitro evidence was presented to justify the potential use of cumulus biomarkers in predicting oocyte competency and IVF outcomes, the feasibility of assessing these biomarkers as an add-on prognostic procedure in IVF is still restricted due to study challenges.

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