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.

Germinal vesicle (GV) oocytes are susceptible to heat stress. However, neither the cellular mechanisms triggered by elevated temperature nor the thermoprotective effects of insulin-like growth factor (IGF) on GV oocytes are completely understood. Therefore, a series of experiments was conducted to determine the direct effects of IGF1 (0, 12.5, 25, 50 and 100ng mL-1) on heat-treated GV oocytes. Butyrolactone-arrested GV oocytes were cultured at 38.5°C (control) or 41°C (heat shock; HS) for 14h in the presence of different concentrations of IGF1. Exposure of GV oocytes to 41°C increased (P<0.05) the number of terminal deoxyribonucleotidyl transferase-mediated fluorescein-dUTP nick end-labelling (TUNEL)-positive oocytes. At concentrations of 12.5 and 25ng mL-1, IGF1 tended to minimise these negative effect of HS (P=0.07). However, neither HS nor IGF1 had any effect on caspase activity. HS also decreased (P<0.05) GV oocyte mitochondrial activity and developmental competence to the blastocyst stage. These deleterious effects of HS were alleviated (P<0.05) by 12.5ng mL-1 IGF1. This concentration of IGF1 did not affect cleavage rate, the percentage of TUNEL-positive blastomeres and total blastocyst cell number regardless of temperature. In conclusion, exposure of GV oocytes to HS triggered the apoptotic cascade and compromised oocyte developmental competence. Physiological concentrations of IGF1 had a beneficial effect on heat-shocked GV oocytes.

The role of insulin-like growth factor 1 (IGF1) on cellular function and developmental capacity of heat-shocked oocytes has not been completely understood. Therefore, the objective of this study was to determine the effect of IGF1 on apoptosis, mitochondrial activity, cytoskeletal changes, nuclear maturation, and developmental competence of bovine oocytes exposed to heat shock. Cumulus-oocyte complexes were submitted to control (38.5 °C for 22 hours) and heat shock (41 °C for 14 hours followed by 38.5 °C for 8 hours) in the presence of 0 or 100 ng/mL IGF1 during IVM. Heat shock increased the percentage of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling)-positive oocyte and reduced oocyte mitochondrial activity. However, addition of 100 ng/mL IGF1 minimized these deleterious effects of temperature. Caspase activity was affected neither by heat shock nor IGF1. Exposure of bovine oocytes to 41 °C during the first 14-hour IVM affected cortical actin localization and microtubule organization at the meiotic spindle and reduced the percentage oocytes that reached the metaphase II stage. However, in the presence of IGF1, cortical actin and percentage of metaphase II oocytes were not different between control and heat-shocked oocytes, suggesting a partial beneficial effect of IGF1. There was no effect of IGF1 on microtubule organization. Heat shock also reduced the percentage of oocytes that reached the blastocyst stage, blastocyst cell number, and increased the percentage of TUNEL-positive blastomeres. However, there was no effect of 100 ng/mL IGF1 on oocyte development to the blastocyst stage and blastocyst quality. Therefore, 100 ng/mL IGF1 prevented some heat shock-induced cellular damage in bovine oocytes but had no effect on oocyte developmental competence. In contrast, a low IGF1 concentration (25 ng/mL) had a thermoprotective effect on oocyte developmental competence to the blastocyst stage. In conclusion, IGF1 prevented part of the damage induced by heat shock on oocyte function. This effect was modulated by IGF1 concentration.

Exposure of bovine oocytes to elevated temperature causes many cellular changes such as increased production of reactive oxygen species and reduced mitochondrial activity. Mitochondrial activity has been shown to be associated with expression of nuclear DNA (nDNA) and mitochondrial (mtDNA) encoded genes. There is evidence that the negative effect of heat shock on the oocyte mitochondrial activity was attenuated by insulin-like growth factor-I (IGF-I) supplementation during in vitro maturation (IVM). Therefore, the objective of this study was to determine the effect of IGF-I on mRNA expression of nDNA (TFAM: mitochondrial transcription factor A and ATP5S: ATP synthase subunit s) and mtDNA (ATP6: ATP synthase subunit 6 and COX1: cytochrome C oxidase subunit 1) encoded genes in bovine oocytes subjected to heat shock during IVM. Cumulus-oocyte complexes (COCs) recovered from slaughterhouse ovaries were distributed in control (22h at 38.5°C) and heat shock (14h at 41°C and 8 hours at 38.5°C) groups in the presence of 0 or 25 ng/mL IGF-I during IVM. After IVM, COCs were mechanically denuded by repeated pipetting for complete removal of cumulus cells. Denuded oocytes were stored at -80°C until RT-PCR. Groups of 30 oocytes per replicate were collected from each experimental group (n = 5 replicates) and submitted to total RNA extraction (RNeasy Mini kit, Qiagen). Reverse transcription (RT) reaction was performed using the Superscript III Kit (Invitrogen). Amplification of target genes was carried out using power SybrGreen® PCR Master Mix. The expression of genes ATP6, COX1, and TFAM ATP5S was determined by real time RT-PCR. Cyclophilin A expression was used as reference gene according to the RefFinder program. Relative gene expression values were obtained by ΔΔCt method corrected by the amplification efficiency for each gene (Pfaffl equation). Data were submitted to least squares analysis of variance using the SAS statistical software. There was no effect of temperature and IGF-I on ATP6, TFAM and ATP5S mRNA expression. Exposure of bovine oocytes to heat shock during IVM increased (Temperature: P < 0.0005; Temperature x IGF: P < 0.01) COX1 mRNA expression as compared to control. However, supplementation of heat shocked oocytes with 25 ng/mL IGF-I during IVM recovered COX1 gene expression to levels similar to the control group. In conclusion, IGF-I has a regulatory action in COX1 gene expression, possibly acting indirectly on the respiratory chain activity under heat shock.

High environmental temperatures observed during the hot months of the year reduce fertility in lactating dairy cows. Summer heat stress depression in fertility is a multifactorial problem that affects physiological and cellular functions in several tissues. It has been shown that in addition to compromise follicular development, hormonal secretion, endometrial and embryonic function, heat stress also markedly reduces oocyte developmental ability. Oocyte susceptibility to elevated temperature can be detected during the germinal vesicle (GV) and oocyte maturation periods. In vivo (heat stress) and in vitro (heat shock) experiments indicated that exposure of bovine oocytes to elevated temperature affects the events required for successful oocyte maturation, fertilization and preimplantation embryonic development. This heat-induced decrease in oocyte function occurs due to a series of cellular alterations that affects nuclear and cytoplasmic compartments of the bovine oocyte. However, thermoprotective molecules such as growth factors and apoptosis inhibitors, which rescue heat-induced oocyte damage and developmental competence, can reverse these cellular changes. Therefore, identification of thermoprotective molecules can be considered as an alternative to modulate the effects of elevated temperature in reproductive function.

Adverse environmental conditions such as elevated temperature and humidity compromise fertility of lactating dairy cows. The series of events associated with oocyte growth and maturation are susceptible to disruption by elevated temperature compromising the ability of the oocyte to undergo adequate fertilization and embryonic development. Such events are regulated by a variety of growth factors and dynamic communication between the oocyte and its surrounding cumulus cells. Previous studies in our laboratory have demonstrated a beneficial effect of insulin-like growth factor-I (IGF-I) reducing heat-induced apoptosis in bovine oocytes exposed to elevated temperature. Therefore, the objective of the current study was to determine the role of IGF-I on developmental competence of bovine oocytes exposed to heat shock. Cumulus-oocyte complexes (COCs) collected from slaughterhouse ovaries were exposed to control (39°C) or heat shock (41°C) treatments in the presence of 0 or 100 ng/ml IGF-I during the first 14 hours in vitro maturation (14h-IVM). Oocytes were subjected to in vitro fertilization and culture at 39°C. The percentage of oocytes that cleaved and developed to the blastocyst stage was evaluated on days 3 and 9 after fertilization, respectively. This experiment was replicated 6 times using 238-253 oocytes/treatment. Day 9 blastocysts were processed for TUNEL (Terminal deoxynucleotidyl transferase nick-end labeling) and Hoechst labeling. Data were analyzed by least-squares analysis of variance using the General Linear Models procedure of SAS. There was no effect of Temperature, IGF-I or Temperature x IGF-I interaction on cleavage rates. However, the percentage of oocytes that reached the blastocyst stage was affected by Temperature (P< 0.05) and IGF-I (P< 0.05). In the absence of IGF-I blastocyst development was reduced from 33.0 to 17.0% for 39 and 41°C, respectively. On the other hand, in the presence of IGF-I blastocyst development was 17.3 versus 16.6% for 39 and 41°C, respectively. In this study blastocyst total cell number was not affected by Temperature, IGF-I or Temperature x IGF-I interaction. However, the percentage of TUNEL-positive blastomeres was affected by Temperature (P< 0.05) and IGF-I (P< 0.05). Heat shock during 14h-IVM increased the percentage of TUNEL-positive blastomeres from 1.0 to 5.0% for 39 and 41°C, respectively. On contrast, IGF-I reduced the proportion of TUNEL-positive blastomeres from 6.0 to 0.0% for 0 and 100 ng/ml IGF-I, respectively. In conclusion, exposure of bovine oocytes to physiological heat shock of 41°C during the first 14h-IVM reduced preimplantation embryonic development and increased the proportion of blastomeres positive to apoptosis. Moreover, supplementation of maturation medium with 100 ng/ml IGF-I exerted a negative effect on subsequent blastocyst development. It is possible that COCs response to IGF-I is modulated by different IGF-I concentrations. (Research supported by FAPESP 2007/53323-0 and 2010/07673-2; CNPq 478558/2008-2). (poster)

The series of events associated with oocyte maturation are susceptible to disruption by elevated temperature. These events are regulated by a variety of growth factors, such as insulin-like growth factor-1 (IGF-1). Exposure of bovine oocytes to heat shock compromises oocyte competence and triggers apoptosis. It has been shown that cellular stresses often alter mitochondrial function and activate the mitochondrial apoptotic cascade. Therefore, the objective of this study was to determine the effect of heat shock on bovine oocyte mitochondrial activity and the role of IGF-1 in this context. Slaughterhouse derived cumulus–oocyte complexes (COC) were subjected to control (38.5°C for 22 h) and heat shock (41°C for 14 h, followed by 38.5°C for 8 h) treatments in the presence of 0 or 100 ng mL–1 of IGF-1 during in vitro maturation (IVM). After 22 h, IVM COC were mechanically denuded and subjected to MitoTracker Red CMX-Ros assay (Invitrogen M-7512) to localize and quantify active mitochondria. Denuded oocytes were incubated in TCM-199-HEPES containing 10 μg mL–1 of polyvinyl alcohol and 50 nM MitoTracker at 37°C for 15 min. Oocytes were evaluated under fluorescence microscope and digital images were obtained and stored as TIFF files. Mitochondrial activity from each oocyte was quantified using the software Image J 1.43. This experiment was replicated 6 times using 97 to 204 COC/treatment. Data were analyzed by least-squares analysis of variance using the general linear model procedure of SAS. In the absence of IGF-1, heat shock reduced (P < 0.001) mitochondrial activity from 64.31 ± 1.91 to 56.74 ± 1.26 arbitrary units for control and heat shock groups, respectively. Addition of IGF-1 to maturation medium did not affect mitochondrial activity in the control group (66.25 ± 1.56). However, IGF-1 improved (temperature × IGF-1; P < 0.001) mitochondrial activity of bovine oocytes subjected to heat shock (70.32 ± 1.32). In conclusion, heat shock reduced bovine oocyte mitochondrial activity, suggesting activation of mitochondrial apoptotic cascade. Moreover, IGF-1 exerted a thermoprotective role, reducing the mitochondrial damage caused by elevated temperature.

Heat-stress induced maternal hyperthermia has been shown to compromise the series of events associated with oocyte growth and maturation reducing oocyte competence. Such events are regulated by a variety of growth factors and dynamic communication between the oocyte and its surrounding cumulus cells. The objective of the current study was to evaluate the modulatory effects of COCs quality and IGF-I on mitochondrial membrane potential (MMP) and apoptosis in cumulus cells induced by heat shock. In this study high (≥3 layers of compact cumulus cells and homogeneous cytoplasm) and low-grade COCs (<3 layers of less compact cumulus cells and irregular cytoplasm) derived from slaughterhouse ovaries were exposed to control (CTR: 39°C) or heat shock (HS: 41°C) treatments in the presence of 0 or 100 ng mL-1 IGF-I during the first 12 h of in vitro maturation (12 h-IVM). Immediately after 12 h-IVM COCs were denuded by repeated pipetting and cumulus cells evaluated for MMP (MitoProbe JC-1 assay kit. JC-1 is a cationic dye that exhibits potential-depend accumulation in the mitochondria) and apoptosis (Annexin V-FITC and propidium iodide) by flow cytometry (Guava EasyCyte Mini Flow Cytometry System, Millipore, Billerica, MA, USA). This factorial experiment was replicated 4 times using 75-100 COCs per treatment. Data were subjected to three-way analysis of variance using the General Linear Models procedure of SAS. Results are shown in Table 1. Exposure of high and low-grade COCs to HS reduced (P < 0.01) the percentage of cumulus cells carrying high MMP regardless of IGF-I. Even though HS caused cumulus cells mitochondrial membrane depolarization there was neither temperature nor COCs quality effect on cumulus cells apoptosis as indicated by the lack of phosphatidylserine (PS) translocation from the inner to the outer leaflet of the plasma membrane. On the other hand, addition of IGF-I to maturation medium reduced (P < 0.05) the percentage of cumulus cells labeled with Annexin V + PI regardless of COCs quality or temperature. There was no statistical interaction between COCs quality × IGF-I × temperature. In conclusion, exposure of COCs to HS during 12 h-IVM caused cumulus cells mitochondrial depolarization without inducing apoptosis. It is possible that a period longer than 12 h is required for most PS translocation to occur in cumulus cells. Moreover, IGF-I exerted protective effect reducing cumulus cells late apoptosis/necrosis events. Table 1.Effect of heat-shock and IGF-I on cumulus cells mitochondrial membrane potential and apoptosis. Results are least-squares means ± SEM.