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To investigate the effects of Insulin like growth factor-1 (IGF-1) on bovine oocyte developmental competence under heat stress.
In Experiment 1, bovine cumulus-oocyte complexes (COCs) were cultured at 38.5 or 41°C for the first 12 h of maturation in the presence of either 100 ng/ml human recombinant (hr)-IGF-1 or acetic acid. In Experiment 2, COCs were cultured in 38.5 or 41°C for the first 12 h of maturation in the presence either of 100 ng/ml hr-IGF-1 or acetic acid. After fertilization, putative zygotes were cultured for 8 days.
In experiment 1, addition of rh-IGF-1 to maturation medium at 38.5°C significantly increased the proportion of M II oocytes and decreased the percentage of TUNEL-positive oocytes compared to the other groups. However, addition of rh-IGF-1 to maturation medium under heat stress increased the percentage of TUNEL-positive oocytes. In experiment 2, addition of rh-IGF-1 under heat sress did not affect cleavage rate, whereas, blastocyst formation rate decreased in heat-stressed and heat-stressed plus rh-IGF-1 groups. Similarly, The number of trophectoderm cells and total cell number were decreased in heat-stressed and heat-stressed plus rh-IGF-1 groups and the percentage of TUNEL-positive nuclei were increased in heat-stressed and heat-stressed plus rh-IGF-1 groups compared to the other groups.
The results of the present study demonstrate that IGF-1 decreases oocyte developmental competence and total cell number and increases TUNEL-positive nuclei at heat stress condition. These unexpected results of IGF-1 during maturation period under heat stress condition warrant further optimizations and investigations.
Embryonic loss is one of the most important factors affecting the reproduction efficiency of high producing dairy cattle [1, 2]. In addition, one of the major factors that lead to embryonic loss and reduced fertility in the hot seasons is heat stress [3, 4]. Heat stress is a worldwide problem, which inflicts heavy economics losses and effect about 60% of the cattle population in the world . Field fertility data in cattle revealed the conception rates of 10–20% in the hot summer vs 50–55% in non-hot seasons . While the exact causes for the lower embryonic viability and reduced conception rates are not yet clear, heat stress-induced damages to oocytes prior to fertilization might contribute, in part, to the low reproductive performance of the cows during the hot seasons. Therefore, improving reproductive efficiency in dairy cattle during warm seasons is a major target in tropical and subtropical areas. Using in vitro trials to study the effects of heat stress on oocyte and embryo make it possible to discover the mechanism by which heat stress exert its effects. Based on in vitro studies, cattle embryo exposure to elevated temperatures at the 2–8 cell stage reduced their developmental competence. However, embryos after maternal to zygote transition are less sensitive to elevated temperatures . There are several studies indicating that oocytes are very susceptible to heat stress . The negative effect of heat stress on developmental competence of the oocyte is mainly due to direct effects of elevated temperature . It has been established that apoptosis is a key event during heat stress of oocytes and embryos under in vitro conditions [7, 9]. Inhibition of apoptosis by means of using well known anti-apoptotic factors such as IGF-1 [9–12] may be a useful tool for decreasing the deterioration effects of heat stress on oocyte developmental competence. Insulin like grow factor-1 is a potent stimulator of oocyte maturation and subsequent embryonic development [13, 14] and is a suppressor of apoptosis [13, 15]. For the current experiments, it was hypothesized that the presence of IGF-1 during in vitro maturation (IVM) as an anti-apoptotic factor can improve bovine oocyte developmental competence under heat stress. Therefore, the aims of this study are to determine the effects of IGF-1 on 1) oocyte nuclear maturation and 2) oocyte developmental competence under heat stress.
Chemicals were purchased from sigma chemical Co. (St. Louis, Mo, USA) and Gibco (Grand Island, NY, USA) unless otherwise indicated.
Bovine embryos were produced by standard in vitro embryo production (IVEP) procedures. Ovaries were obtained from two local abattoirs and transferred in physiological saline containing 100 μg/ml streptomycin and 100 IU/ml penicillin G at 30–35°C within 2 h after collection. At the laboratory, the COCs were aspirated from transparent follicles (2–8 mm in diameter). Only COCs with a homogenous cytoplasm and at least 3 compact layers of surrounding cumulus cells were used. Follicle aspiration medium was based on H-TCM supplemented with 10 mg/ml BSA and 50 IU/ml heparin. Oocytes maturation medium (OMM) consisted of TCM-199 supplemented with FSH (10 μg/ml), LH (10 μg/ml), 17-ß-estradiol (1 μg/ml), and BSA (10 mg/ml) and 100 ng/ml rh-IGF-1 (Upstate Biotechnology, Lake Placid,NY) or 0.1 M acetic acid as a vehicle. After selection, COCs were washed four times (in OMM without hormones and OMM) and matured in groups of 20 in 50-μl drops of OMM overlaid with mineral oil for 24 h at 38.5 or at 41°C during first 12 h of maturation followed by 12 h at 38.5°C in atmosphere of 5% (v/v) CO2 in humidified air (Labotect C200, Germany). After 24 h of incubation in the maturation medium, COCs were washed four times in the Fert-TALP  medium. The Fert-TALP medium supplemented with penicillamine (20.µM), hypotaurine (10µM), epinephrine (1µM) and heparin (0.56µg/ml) was used for fertilization and sperm from the same ejaculation was used throughout these experiments. The frozen sperm was thawed in air and in a warm water bath for 10 and 30 s, respectively and then separated on a discontinuous Pure Sperm gradient (80 and 40%). Sperm at a concentration of 1.5×106/ml and COCs were co-cultured in groups of 40 in 200 μL Fert-TALP medium in a humidified environment of 5% O2, 5% CO2, and 90% N2 at 38.5°C. After 18–20 h post insemination (pi) putative zygotes were stripped of cumulus by vortexing and washed three times in the washing medium. Then, putative zygotes were transferred to SOFaa overlaid with mineral oil, and cultured (1 embryo/μl SOF) in a humidified environment of 5% O2, 5% CO2, and 90% N2 at 38.5°C (Labotect C200, Germany). Three days after fertilization, cleaved zygotes were transferred to fresh SOFaa (supplemented with glucose). Medium refreshes were done on Day 5 and 7 pi.
At the end of maturation period, oocyte nuclear maturation was determined by DAPI-staining as described in details earlier . Briefly, COCs were denuded by vortexing for 3–7 min and then fixed for 15 min in 2.5% (w/v) glutaraldehyde, washed with PBS, stained with 2.5%(w/v) 4,6-diamino-2-phenyl-indole (DAPI), and mounted on glass slides. The nuclear status of the stained oocytes was assessed under a fluorescence microscope (Olympus, BX 51).
The TUNEL assay was used to detect DNA fragmentation associated with late stages of the apoptosis cascade. The in situ cell death detection kit was obtained from Roche Diagnostics Corporation (Indianapolis, IN). The method used for TUNEL labeling was described in details earlier . Briefly, Oocytes and embryos were removed from culture medium, washed three times in 100-μl PBS containing 1 mg/ml PVP (PBS-PVP), fixed in 4% (w/v) paraformaldehyde in PBS for 1 h at room temperature, and stored in PBS-PVP at 4°C. The TUNEL assay was initiated by permeabilizing the oocytes or embryos in 100-μl drops of 0.1% (v/v) Triton X-100 containing 0.1% (w/v) Na-citrate in PBS for 30 min at room temperature. Samples were then incubated in 50-μl drops of TUNEL reaction mixture for 1 h at 37°C in the dark. Oocytes and embryos were then washed in PBS-PVP, transferred to 50 μl drops of 50 μg/ml Propidium iodide (PI) in PBS-PVP for 30 min at room temperature, washed three times in PBS-PVP, placed in a glycerol drop on slide and coverslips mounted. TUNEL labeling was observed using a fluorescence microscope (Olympus, BX 51). Each oocyte was analyzed for TUNEL-positive nucleus and each embryo was analyzed for total number of nuclei and the number of TUNEL-labeled nuclei.
At the end of the culture period, the numbers of cells in expanded blastocysts in all groups were determined by differential staining as described in details earlier . Briefly, blastocysts were first incubated in 500µl of 1% tritonX-100 and 100µg/ml PI for up to 30 s and then immediately transferred into 500µl of 100% ethanol with 25µg/ml bisbenzimide (Hoechst 33258) and stored at 4°C overnight. Fixed and stained embryos were then mounted on a glass slide in a drop of glycerol, gently flattened with a cover slip and visualized for cell counting on a fluorescence microscope.
The experiment was designed with a 2×2 factorial arrangement of treatments. The experiment was performed to examine the effect of heat stress (41°C) and IGF-1 on nuclear maturation and apoptosis of oocytes. Cumulus-oocyte complexes were matured in the presence of 100 ng/ml hr-IGF-1 or similar volume of acetic acid (Table 1) at 38.5°C for 24 h or 41°C for 12 h followed by 38.5°C for 12 h. Oocyte nuclear status and incidence of apoptosis were recorded in all groups 24 h after maturation.
The experiment was designed with a 2×2 factorial arrangement of treatments. Treatments were similar to those in the experiment 1. After in vitro maturation, fertilization and culture were at 38.5°C (Table 2). Cleavage rate and blastocysts formation rate were recorded on Days 3 and 8 post insemination, respectively. Expanded blastocysts were subjected to either TUNEL analysis or differential staining.
Data were analyzed by GLM procedure of SPSS and Duncan’s test for mean differences (P<0.05). Percentage data were transformed using the arcsine transformation before analysis. Data are presented as mean ± standard error of the mean (S.E.M.). The statistical model was Yijk = μ + Hi + Ij + (H × I)ij + eijk, where μ = constant, Hi=heat stress regimes (i=38.5 or 41°C), Ij = IGF-1 levels (j=0 or 100 ng/ml), (H × I)ij = interaction between heat stress regimes and IGF-1 levels, and eijk = error term.
Heat stress at 41°C did not alter the percentage of metaphase II oocytes in the presence or absence of IGF-1, but the presence of IGF-1 in the control temperature (38.5°C) group increased (P<0.05) the percentage of metaphase II oocytes (Fig. 1). The percentage of oocytes that were TUNEL-positive was higher (P<0.05) in oocytes exposed to 41°C for the first 12 h of maturation in presence of IGF-1 than that in the other groups (Fig. 2).
Cleavage rate did not affected by heat stress and IGF-1 treatment (P>0.05), but blastocyst formation rate was significantly (P<0.05) reduced in heat stress and heat stress plus IGF-1 groups compared to normal temperature and normal temperature plus IGF-1 groups (Table 3). Number of inner cell mass (ICM) and trophectoderm (TE) cells and total cell number were affected by different treatments (Figs. 3, ,4,4, and and5).5). Number of ICM and TE cells and total cell number were the lowest (P<0.05) in heat stress plus IGF-1 group. Similarly, TUNEL-positive nuclei ratio in heat stress and heat stress plus IGF-1 groups was higher (P<0.05) than that in normal temperature and normal temperature plus IGF-1 groups (Fig. 6).
Exposure of superovulated cows to direct solar radiation in the summer at day 1 following estrus compromised continued development of embryos when collected on day 8, but exposure to heat stress on days 3, 5, or 7 did not affect embryonic development . This implies that the maturing oocyte is more sensitive to heat stress than embryo as it progresses through development. In the experiment 1, we investigated the role of heat stress and IGF-1 on apoptosis rate and proportion of M II oocytes after in vitro maturation period. In this study, proportion of M II oocytes were significantly (P<0.05) increased by IGF-1 supplementation at normal temperature condition (38.5°C) compared to the other groups. Stimulatory effect of IGF-1 on oocyte maturation and further embryo development is well established [14, 20]. Also, it has been established that IGF-1 has a beneficial effect on oocyte nuclear maturation [21, 22], whereas, oocyte expresses IGF-1 receptor . Forthermore, IGF-1 stimulates resumption of meiosis and completion of nuclear maturation of porcine oocytes . It has been detected that exposure of bovine oocytes to EGF and IGF-1 accelerated progression of meiosis in COCs . The beneficial effect of IGF-1 on oocyte has been proven in vivo. It has been shown that intraovarian injection of IGF-1 in calves improves oocyte developmental competence . In the current study, heat stress and heat stress plus IGF-1 treatments failed to alter the percentage of M II oocytes. This results is not in agreement with the finding of Lenz et al. , they reported that heat stress in in vitro reduced nuclear maturation rate. Probably, this discrepancy is due to a difference in heat stress level. Additionally, heat stress during bovine oocyte maturation reduced the proportion of oocytes that progressed to metaphase II and heat-stressed oocytes often possessed misshapen metaphase I spindles with disorganized microtubules and unaligned chromosomes, indicating that heat stress can disrupt the microtubular network . Also, in this study, the percentage of apoptosis in oocytes was decreased by IGF-1 in normal temperature condition compared to the other groups. This result indicates that IGF-1 act as anti-apoptotic factor during oocyte in vitro maturation under normal temperature condition . Wasilak and Bogacki  suggested that IGF-1 can inhibit apoptosis in oocyte at the stage of caspase activation and consequently prevents further advancement of apoptosis and terminal DNA fragmentation in oocyte. In an experiment, heat stress regime at 40°C or 41°C for the first 12 h of maturation increased percentage of TUNEL-positive oocytes at the end of maturation . Roth and Hansen , also included that heat stress during maturation can promote an apoptotic response mediated by group II caspase. In our study, heat stress at 41°C for the first 12 h of maturation could not significantly decrease the percentage of TUNEL-positive oocytes at the end of maturation. This result is not in agreement with previous experiment by Roth and Hansen . It seems that this conflict come from the diverse response of genotypes in different studies. One of the most interesting finding in this study is the significant increase in the percentage of TUNEL-positive oocytes in heat stress plus IGF-1 group. It seems that simultaneously effect of IGF-1 and heat stress on oocyte activates some unknown cell pathways that result in increase of apoptosis incidence in oocyte. However, we have no clear evidence for this negative effect. Further studies to evaluate the effects of IGF-1 and heat stress at the molecular level in oocyte are needed.
In experiment two, cleavage rate was not affected by heat stress and IGF-1 during maturation period. These data are in agreement with previous studies [11, 30, 31]. However, it seems that cleavage rate is not a sufficiently reliable indicator of developmental potential . In this study, it has shown that IGF-1 failed to change percentage of day 8 blastocyst. These results are in agreement with an experiment from Makarevich and Markkula  and differ from previous results from the same authors , in which IGF-I added to IVM medium increased blastocyst yield. This discrepancy may be caused by a difference in genotype, technical factor, and season that can influence the ability of bovine oocytes to develop to the blastocyst stage . Also, differences between the current study and the others studies may be related to differences in culture condition. It may be that IGF-1 is more effective on blastocyst development when the culture condition is not in optimal . In this study, the percentage of day 8 blastocyst was decreased by heat stress at the first 12 h of maturation. Previous experiment have shown that heat stress during oocyte in vitro maturation results in retarded embryo development  and reduction in blastocyst formation rate [7, 30, 31, 37]. It is thought that heat stress can compromise embryonic development when occurring during maturation. It seems that heat stress during maturation induces some cellular damages that are carried over to the embryonic stage. The mechanisms for this are not exactly defined but heat stress on maturing oocytes appears to impair protein synthesis, and it seems that heat stress can produce free radicals which subsequently they can change the surface of the zona pellucida and cytoplasm  impairing sperm penetration. Also, it has been suggested that heat stress during maturation upregulates apoptotic pathways in bovine oocytes which is a crucial event for the loss of developmental competence thereafter . One of the unexpected results in the experiment two was decrease of blastocyst formation rate in the simultaneously presence of IGF-1 and heat stress. It seems that decrease of blastocyst formation rate is due to increase of apoptosis incidence in oocytes in this group. Also, there is a correlation between increase of glucose consumption and production of ROS though Krebs cycle. On the other hand, it has been established that apoptosis is increased by high glucose concentration in the culture medium and it seems that high temperature and IGF-1 increase glucose consumption more than optimal level and consequently increase incidence of apoptosis. Results of the current study shown that heat stress during maturation induces apoptotic events in bovine oocytes and that the activation of these processes of a critical phenomenon for the loss of developmental competence of the oocyte following heat stress . The total cell number increased after addition of IGF-1 to the IVM medium. The increase in the TUNEL index in heat stress and heat stress plus IGF-1 groups could, at least partially, be explained by the decrease in total cell number in blastocysts in these two groups. The addition of IGF-I to IVM medium under normal temperature condition did not reduce the percentage of TUNEL positive cells. In this study, number of trophectoderm cell per blastocyst was decreased by heat stress and heat stress plus IGF-1 groups. The number of ICM cells decreased in heat stress plus IGF-1 group compared to the other groups. Therefore, the reduced blastocyst total cell number was mainly due to a decrease in the trophectoderm (heat stress group) and TE and ICM (heat stress plus IGF-1 group). These data are in agreement with the previous study by Ju et al. . It is intriguing to consider that heat stress with or without IGF-1 during maturation period exerted more deleterious effect on subsequent trophectoderm cells. On the contrary, it has been reported that mouse ICM cells were more sensitive to heat stress at the blastocyst stage . Meanwhile, TE cells can produce bIFN-τ  for maternal recognition of pregnancy (MRP) during early pregnancy . Secretion of bIFN-τ will be reduced during heat stress condition . Therefore, the decrease of trophectoderm cell number in response to heat stress is likely associated with the post-implantation pregnancy losses during hot seasons . Also, in one experiment that has been carried out by Heyman et al. , data have shown that co-transfer of frozen bovine blastocyst and trophoblastic vesicles increase pregnancy rate in recipients. This result shows that trophectoderm has an important role in the implantation and establishment of pregnancy. On the other hand, in some reports the removal of a part of trophectoderm can not decrease pregnancy. It seems that removal a little part of trophectoderm has no a serious detrimental effect on pregnancy rate compared to effects of freezing and heat stress.
Finally, recombinant bovine somatotropin (bST) can stimulate IGF-1 secretion in many tissues including reproductive tract. If we can find out the best time for positive effect of IGF-1 on bovine embryo development under heat stress, it could be suggested that administration of bST in that time probably will have a beneficial effect on pregnancy rate via increase of local IGF-1 in dairy cattle during the hot season.
In conclusion, this study indicates that IGF-1 plays a detrimental role on bovine oocyte developmental competence under heat stress condition in vitro. It is thought that IGF-1 decreased bovine oocyte developmental competence via increased apoptosis in oocytes under heat stress conditions. From these results, further studies are needed to investigate the exact mechanisms of this effect.
This study was funded by Royan institute of Iran, grant number 86/31684. The authors would like to thank University of Tehran and M. Hajian and L. Hosseini from the laboratory of the Royan institute, Isfahan campus for their technical supports.
Capsule Bovine oocyte developmental competance is decreased by simultaneously effect of IGF-1 and heat stress.