Activation Of Bovine Oocytes Research Articles

Bovine oocyte activation with bull or human sperm by conventional ICSI and Piezo-ICSI: Its relationship with PLCɀ activity.

The intracytoplasmic sperm injection (ICSI) technique has low efficiency in cattle. This has mainly been attributed to the oocyte activation failure due to oocyte and/or sperm factors. Our aim was to evaluate the effect of conventional ICSI and Piezo-ICSI with bull or human sperm on bovine oocyte activation and embryo development and to assess its relationship with the phospholipase C zeta (PLCɀ) activity of both species. In vitro matured bovine oocytes were randomly divided into five groups and were fertilized as follows: conventional ICSI using bovine sperm with chemical activation (control), conventional ICSI using bovine sperm, Piezo-ICSI using bovine sperm, conventional ICSI using human sperm, and Piezo-ICSI using human sperm. PLCɀ activity was determined in bull and human sperm samples. Within the groups using bull sperm, the oocytes fertilized by conventional ICSI had the lowest values of 2 pronuclei (PN) formation and cleavage, Piezo-ICSI increased both percentages and ICSI + chemical activation presented the highest 2 PN, cleavage, and blastocyst rates (p < 0.05). Within the groups using human sperm, the oocytes fertilized by Piezo-ICSI presented higher 2 PN and cleavage rates than those activated by conventional ICSI (p < 0.05). Piezo-ICSI with human sperm increased bovine oocyte activation as much as conventional ICSI + chemical activation with bovine sperm (p < 0.05). Higher values of PLCɀ activity were found in human sperm compared with bovine sperm (p < 0.05). Our results suggest that the higher stability of the bovine sperm in combination with its relatively low content of PLCɀ impairs bovine oocyte activation after ICSI.

35 Koala sperm induces bovine oocyte activation after intracytoplasmic sperm injection

35 Koala sperm induces bovine oocyte activation after intracytoplasmic sperm injection

Combined Exogenous Activation of Bovine Oocytes: Effects on Maturation-Promoting Factor, Mitogen-Activated Protein Kinases, and Embryonic Competence.

Oocyte activation via dual inhibition of protein synthesis and phosphorylation has improved in vitro embryo production in different mammalian species. In this study, we evaluated the effects of the combination of cycloheximide (CHX), dimethyl amino purine (DMAP), and anisomycin (ANY) on the activation of bovine oocytes, particularly on dynamics of MPF and MAPKs, embryonic developmental potential, and quality. The results showed that the cleavage and blastocyst rates, as well as levels of CCNB1, CDK1, p-CDK1Thr161, and p-CDK1Thr14-Tyr15, were similar among groups; ANY and ANY + CHX reduced the expression of ERK1/2 compared to DMAP-combinations (p < 0.05), whereas ANY + DMAP, CHX + DMAP, and ANY + CHX + DMAP reduced p-ERK1/2 compared to ANY and ANY + CHX treatments (p < 0.05). The quality of blastocysts in terms of cell counts, their allocation, and the numbers of TUNEL-positive cells did not differ among groups. However, transcript levels of POU5F1 were higher in embryos derived from ANY + CHX + DMAP treatment compared to other groups, while expression levels of CDX2 did not show differences. In addition, the BCL2A1/BAX ratio of the ANY + CHX + DMAP treatment was significantly low compared to the ANY treatment (p < 0.05) and did not differ significantly from the other treatments. In conclusion, oocyte activation by dual inhibition of protein synthesis and phosphorylation induces MPF inactivation without degradation of CCNB1, while MAPK inactivation occurs differentially between these inhibitors. Thus, although the combined use of these inhibitors does not affect early developmental competence in vitro, it positively impacts the expression of transcripts associated with embryonic quality.

Influence of calcium ionophore on the fertilization of bovine oocytes and their further embryonic development

Intracytoplasmic spermatozoid injection (ICSI) is one of the commonest methods used in assisted reproductive technologies in human medicine. However, this procedure has low efficacy for bovines, mainly because of insufficient activation of oocytes after spermatozoid microinjection. One of the most effective methods of activating oocytes is considered to be the use of phosphorus calcium, though the optimal concentration of activator and its effect on pre-implant development of embyo are still open questions. An oocyte-cumulus complex of clinically healthy cows, retrieved from the ovaries during slaughter, matured over 22–24 h in in vitro conditions. Oocytes with visible polar body had been subjected to intracytoplasmic spermatozoid injection (ICSI), and were 15–30 min later activated in the environment with different concentrations of calcium ionophore for 15–20 min and then transferred for further cultivation in a culture medium with sodium pyruvate. The fertilization rate was identified on the second day at the 2–4th stages of cellular embryo, and the quality of obtained embyos was evaluated on day 8. Based on the statistical analysis of the data, we determined that the artificial activation of bovine oocytes using calcium ionphore after intracytoplasmic spermatozoid injection (ICSI) led to statistically significant improvement in conception and ratio of blastocytes obtained to oocytes injected. In the study, we confirmed that addition of 5, 10 and 50 µМ of the agent had the same efficacy on the activation of occytes of bovine cattle. However, it has to be noted that during further cultivation of the obtained zygotes up to the blastocyte stage (day 8), we saw no significant differences in quality of embryos obtained. Therefore, use of calcium ionophore for the activation of bovine oocytes after intracytoplasmic spermatozoid injection is effective, for it promotes increase in fertilization parameters and ratio of blastocytes obtained to oocytes injected, facilitating production of higher numbers of embyos suitable for transplantation or cooling. Our previous conclusions are valuable for increasing the efficacy of methods of intracytoplasmic injection of bovine spermatozoid and its further use for purposes of science and production.

138 Combination of protein synthesis and phosphorylation inhibitors on the activation of bovine oocytes

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

139 Exogenous activation of bovine oocytes after piezo-intracytoplasmic sperm injection: is it really necessary?

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Activation of bovine oocytes by protein synthesis inhibitors: new findings on the role of MPF/MAPKs†.

The present study evaluated the mechanism by which protein synthesis inhibitors activate bovine oocytes. The aim was to analyze the dynamics of MPF and MAPKs. MII oocytes were activated with ionomycin (Io), ionomycin+anisomycin (ANY) and ionomycin+cycloheximide (CHX) and by in vitro fertilization (IVF). The expression of cyclin B1, p-CDK1, p-ERK1/2, p-JNK, and p-P38 were evaluated by immunodetection and the kinase activity of ERK1/2 was measured by enzyme assay. Evaluations at 1, 4, and 15hours postactivation (hpa) showed that the expression of cyclin B1 was not modified by the treatments. ANY inactivated MPF by p-CDK1Thr14-Tyr15 at 4hpa (P<0.05), CHX increased pre-MPF (p-CDK1Thr161 and p-CDK1Thr14-Tyr15) at 1hpa and IVF increased p-CDK1Thr14-Tyr15 at 17hours postfertilization (hpf) (P<0.05). ANY and CHX reduced the levels of p-ERK1/2 at 4hpa (P<0.05) and its activity at 4 and 1hpa, respectively (P<0.05). Meanwhile, IVF increased p-ERK1/2 at 6hpf (P<0.05); however, its kinase activity decreased at 6hpf (P<0.05). p-JNK in ANY, CHX, and IVF oocytes decreased at 4hpa (P<0.05). p-P38 was only observed at 1hpa, with no differences between treatments. In conclusion, activation of bovine oocytes by ANY, CHX, and IVF inactivates MPF by CDK1-dependent specific phosphorylation without cyclin B1 degradation. ANY or CHX promoted this inactivation, which seemed to be more delayed in the physiological activation (IVF). Both inhibitors modulated MPF activity via an ERK1/2-independent pathway, whereas IVF activated the bovine oocytes via an ERK1/2-dependent pathway. Finally, ANY does not activate the JNK and P38 kinase pathways.

Endoplasmic reticulum distribution during bovine oocyte activation is regulated by protein kinase C via actin filaments.

Fertilization-induced [Ca2+ ]i oscillations generally depend on the release of calcium ions from the endoplasmic reticulum (ER). Since ER is the main store of calcium ions, it plays an important role in oocyte fertilization. However, the mechanism of ER organization at oocyte activation is unknown. Here, we show that protein kinase C (PKC) is involved in ER distribution during bovine oocyte activation, but not involved in cell cycle resumption and spindle organization. Actin filaments were affected by PKC pharmacological inhibition. In addition, similar to PKC results, the actin-depolymerizing drug cytochalasin B affected the ER distribution during oocyte activation. Specifically, we have demonstrated that ER organization during bovine oocyte activation is regulated by PKC possibly through its action on actin filaments regulation. Taken together, the results presented here provide further information on the pathway involved in the regulation of ER organization during oocyte activation and new insight into the functional role of PKC and actin filaments during this process.

Effect of single and combined treatments with MPF or MAPK inhibitors on parthenogenetic haploid activation of bovine oocytes

In bovine, correct oocyte artificial activation is a key step in ICSI and other reproductive biotechnologies, and still needs to be improved. The current study was designed to compare the activating efficiency of ionomycin (Io) followed by: a 4 h time window and ethanol (4h-Et), roscovitine (Rosc), dehydroleucodine (DhL), cycloheximide (CHX) or PD0325901 (PD), each as a single treatment, and then combine them in novel protocols. Parthenogenetic haploid activation was evaluated in terms of pronuclear (PN) formation, second polar body (2PB) extrusion, ploidy of day 2 embryos and in vitro development. Combined treatments with Io-4h-Et-Rosc and Io-Rosc/CHX increased PN formation (92.2% and 96%, respectively) compared with Io-Rosc, Io-CHX or Io-4h-Et, which were equally efficient at inducing PN formation (82–84%) and 2PB extrusion (62.1–70.5%). Oocyte activation with Io-DhL and Io-Rosc/DhL resulted in higher 2PB extrusion rates (90% and 95.9%, respectively) but lower PN formation (49.4–58.8%) and cleavage rates (36–57.9%), as occurred with Io-CHX/DhL (76.4% and 70.4%, respectively). For the first time, results show that Io followed by the MAPK inhibitor PD induces PN formation and 2PB extrusion, but PD combined with Rosc or CHX resulted in low rates of haploid day 2 embryos. In conclusion, DhL strongly induces 2PB extrusion but leads to poor PN formation and embryo development. PD induces bovine oocyte activation but results in low rates of haploid embryos. In contrast, the improved PN formation rates after treatment with combined Io-4h-Et-Rosc and Io-Rosc/CHX suggest they should be further evaluated in ART, aiming to increase success rates in bovine.

152 Activation of Bovine Oocytes Using the Zinc Chelator TPEN

Traditionally, artificial oocyte activation has been induced by stimulating intracellular calcium increase in the oocyte. Recently, the use of zinc chelators has also shown to be effective in inducing activation by decreasing intracellular concentration of zinc, mimicking events during fertilization; however, this has not been demonstrated in bovine oocytes. The use of artificial activation in bovine has potential for overcoming subfertility-related production loss and aid in livestock cloning. In this study, we determined whether bovine oocytes could be artificially activated in the presence of the zinc chelator TPEN [N,N,N’,N’-tetrakis(2-pyridylmethyl) ethane-1,2-diamine]. Bovine cumulus–oocyte complexes (COC) were collected from abattoir-derived ovaries and incubated for 24 h in TCM-199 maturation medium supplemented with fetal bovine serum, sodium pyruvate, Glutamax, oestradiol, and FSH. The COC were denuded by vortexing in denuding medium containing 0.1% hyaluronidase, and individual oocytes were selected based on presence of a visible polar body. Matured oocytes were then incubated in TL-HEPES medium supplemented with 1 of 5 treatments for parthenogenetic activation: (1) DMSO for 2 h (control, n = 116), (2) 100 µM TPEN for 45 min (100-45, n = 103), (3) 100 µM TPEN for 120 min (100-120, n = 102), (4) 200 µM TPEN for 45 min (200-45, n = 63), or (5) 200 µM TPEN for 120 min (200-120, n = 142). After treatment, oocytes were washed with culture media and incubated in droplets of SOF-Be1 medium under oil to monitor subsequent development. The number of blastocysts was recorded on Day 10 of culture. Blastocysts were stained with Hoechst for 15 min to evaluate total cell number. The frequencies of blastocyst formation were compared using the Chi-squared test, and differences in total cell number were compared using the Student’s t-test. All TPEN treatments significantly increased the number of oocytes developed to the blastocyst stage relative to the control group, which was unable to form blastocysts (P &lt; 0.01). The 100-45 treatment had a greater % blastocysts compared with the 200-120 treatment (16.5% vs 7.77%; P &lt; 0.05), tended to be greater than 100-120 treatment (16.5% vs 7.8%, P = 0.058), and was numerically greater than the 200-45 treatment (16.5% vs 7.94%; P = 0.114). Three treatments that resulted in blastocysts were analyzed for cell counting: 200-120 (n = 5), 100-120 (n = 4), and 100-45 (n = 7). Average total cell number was 119.20 ± 52.28 for the 200-120 group, 83.75 ± 51.06 for the 100-120 group, and 111.71 ± 59.06 for the 100-45 group. There was no difference in total cell number among groups (P ≥ 0.341). Here, we demonstrated that mature bovine oocytes can successfully be parthenogenetically activated by incubating with the zinc chelator TPEN. Oocytes incubated with 100 µM TPEN for 45 min provided the greatest blastocyst yield. Total cell number did not differ between treatments, but all groups analyzed showed blastocysts containing over 100 cells, demonstrating the effectiveness of the oocyte activation approach. Further studies will focus on optimizing the use of TPEN to activate bovine oocytes.

188 HAPLOID ACTIVATION OF BOVINE OOCYTES WITH IONOMYCIN AND SINGLE OR COMBINED ACTIVATING AGENTS

Haploid activation of bovine oocytes is important for reproductive technologies such as intracytoplasmic sperm injection (ICSI) or somatic cell nuclear transfer (SCNT). Nevertheless, it is still a highly inefficient procedure. The aim of this work was to combine different activation drugs, known to have different targets along the activation cascade, to find a more effective activation protocol. Cumulus-oocyte complexes (COC) were aspirated from slaughtered ovaries and in vitro-matured (IVM) for 22 h. Oocytes were activated with 5 µM ionomycin (IO) for 4 min and then randomly allocated into 1 of the following treatments: 50 µM roscovitine (ROSC), 10 µg mL–1 cycloheximide (CHX), ROSC and 10 µM PD0325901 (ROSC/PD), or CHX and PD (CHX/PD) for 5 h; 15 µM dehydroleucodine (DHL) or DHL and ROSC (DHL/ROSC) for 3 h; DHL and CHX for 3 h followed by 2 h with CHX; 5-min exposure to 7% ethanol 4 h post-IO (ET); or ET followed by ROSC (ET-ROSC). Controls were IO followed by 3 h of exposure to 1.9 mM 6-DMAP with or without a previous 3-h culture in TCM-199 (3 h in DMAP and DMAP, respectively). Embryos were cultured in SOF medium. Pronuclear formation (PN) and second polar body extrusion (2PB) were assessed by 5 µg mL–1 propidium iodide oocyte staining, 17 h after IO. Activation was defined as the presence of at least 1 PN, and 2PB extrusion rate was calculated regardless of the nuclear stage. Data were analysed by Fisher’s Test (P &lt; 0.05). Activation (Table 1) was similar in all groups, with the exception of ROSC/PD and ET-ROSC that were the highest and DHL that was the lowest. Although ROSC or CHX seemed to improve 2PB rate when combined with DHL, cleavage decreased significantly, suggesting DHL itself, or its combination with these drugs, negatively affects embryo development. Group ET showed activation rates comparable to other treatments, but it was not reflected on cleavage, suggesting that ET induces PN formation but it might be inefficient to trigger embryo development. Nevertheless, this observation was not made for ET-ROSC, as it showed a higher cleavage rate than ET and ROSC alone. The mitogen-activated protein kinase (MAPK) inhibitor PD showed different effects when combined with ROSC or CHX, despite that they both act on the mammary fat pad (MPF). In ROSC/PD, a slight improvement was observed on activation and cleavage rates compared with ROSC. Group CHX/PD resulted in a slightly higher 2PB percentage, but a lower activation percentage that derived in a significantly lower cleavage than CHX. In conclusion, ROSC and CHX were the most effective single treatments for haploid activation. Moreover, some combined treatments, namely DHL/ROSC and DHL/CHX, proved to be as effective or better at 2PB extrusion rate, which is the defining feature in haploid activation. Table 1.Activation, second polar body extrusion (2PB) and cleavage of bovine oocytes activated with ionomycin followed by single or combined activating agents1

Activation treatment of recipient oocytes affects the subsequent development and ploidy of bovine parthenogenetic and somatic cell nuclear transfer (SCNT) embryos.

We evaluated the potential effect of anisomycin, an antibiotic produced by Streptomyces griseolus, on the parthenogenetic activation of bovine oocytes and reconstructed somatic-cell nuclear transfer (SCNT) embryos. A higher cleavage and blastocyst rate were achieved with anisomycin (70.3% and 27.8%) and 6-dimethylaminopurine (DMAP) (73.3% and 30.2%), relative to oocytes parthenogenetically activated with cycloheximide (CHX) (54.1% and 20.2%). In reconstructed SCNT embryos, a greater proportion of embryos reached the blastocyst stage after anisomycin (32.2%) compared to DMAP (22.3%) and CHX (23.5%) treatment. Furthermore, the quality of embryos-assessed by the total number of cells and the inner cell mass-to-total-cell ratio-was higher with anisomycin (166.2 ± 6.9 and 26.9 ± 1.9) compared to DMAP (135.0 ± 8.7 and 39.4 ± 3.5) and CHX (149.1 ± 8.4 and 36.3 ± 2.5), while a lower percentage of chromosomal abnormalities was observed with anisomycin compared to DMAP and CHX treatments, both in parthenotes (though not significant) and in SCNT embryos (P < 0.05). Therefore, anisomycin can enhance the in vitro developmental potential in parthenotes and reconstructed SCNT embryos, specifically improving the quality of SCNT embryos and decreasing the abnormal ploidy of parthenotes and SCNT embryos compared to the traditional protocols of chemical activation with DMAP and CHX. These results may have important implications for the success of reproductive technologies, including SCNT and intracytoplasmic sperm injection, in different mammalian species.

320 EFFECTS OF DIFFERENT ACTIVATION REGIMENS ON PRONUCLEAR FORMATION AND PRE-IMPLANTATION DEVELOPMENTAL COMPETENCE OF IN VITRO-MATURED BOVINE OOCYTES AFTER INTRACYTOPLASMIC SPERM INJECTION

Intracytoplasmic sperm injection (ICSI) is an assisted reproductive technique that has been used with considerable success in humans; however, in the bovine species the efficiency of this technique is far from optimal. The objective of the present study was to evaluate the effect of 4 chemical activation treatments, 6-dimethylaminopurine (DMAP), cycloheximide (CHX), anisomycin (ANY), and ethanol (EtOH) on the pronuclear formation and embryo development of bovine embryos generated by ICSI. Cumulus-oocyte complexes were aspirated from abattoir ovaries, selected, and matured in 400-µL drops of standard TCM-199 maturation medium for 22 h at 38.5°C and 5% CO2. The ICSI was performed by a standard procedure. Injected oocytes were randomly distributed and activated by 5 µM ionomycin for 5 min (Io) followed by i) 5 µg mL–1 CHX for 5 h (Io/CHX), ii) 3 h window followed by a second Io treatment plus 1.9 mM DMAP for 4 h (2Io/DMAP), iii) 1 µg mL–1 ANY for 5 h (Io/ANY), and iv) 3 h window followed by 7% ethanol (Io/EtoH). Embryos were cultured in 50-µL drops of KSOM medium under mineral oil at 38.5°C and 5% CO2, 5% O2, and 90% N2. Cleavage was recorded at 72 h and blastocyst rate at 192 h. Pronuclear formation analysis was carried out at 18 hpa with Hoechst staining. An oocyte was considered fertilized when 2 polar bodies and 1 female and 1 male pronucleus (or a decondensed sperm head) could be observed. The data were transformed to arcsine, analysed by ANOVA, and means were compared using Tukey's test with Statgraphics Plus 2 Software. Results with a total of 431 injected oocytes (114, 104, 101, and 112 for DMAP, CHX, ANY, and EtOH, respectively) showed differences in cleavage (P &lt; 0.01) in DMAP, CHX, and ANY treatments (86, 72, and 78%, respectively), relative to EtOH (12%). Similarly, the rate of blastocysts/injected oocyte at 192 h was higher with DMAP, CHX, and ANY (41, 20, and 32%, respectively), relative to EtOH (4%). Sham-injected oocytes showed cleavage and blastocyst rates of 67, 43, 68, and 12% and 32, 11, 19, and 5%, for DMAP, CHX, ANY, and EtOH, respectively. Despite the higher developmental rate observed with DMAP, pronuclear formation assessment revealed that fertilization rate was higher in CHX (87%) and ANY (75%) treatments relative to DMAP (35%). In conclusion, the results of the present study show that activation of bovine oocytes after ICSI is more efficient with DMAP and ANY, compared with CHX and EtOH.Provision of ovaries by our local slaughterhouse (Frigorifico Temuco, Chile) and funding support from FONDECYT 1120241 CONICYT, Chile, are gratefully acknowledged.

273 EFFECT OF IONOMYCIN ASSOCIATED WITH ROSCOVITINE, DEHYDROLEUCODINE, CYCLOHEXIMIDE, OR ETHANOL ON HAPLOID ACTIVATION OF BOVINE OOCYTES

Haploid activation of bovine oocytes after ICSI is a routine procedure. However, embryos frequently contain an abnormal chromosome set as a result of the drugs employed. We compared the efficiency of ionomycin (Io) followed by roscovitine (ROSC), cycloheximide (CHX), ethanol, and dehydroleucodine (DhL) to induce haploid parthenogenetic activation in bovine. Pronuclear (PN) formation, second polar body (2PB) extrusion, embryo development, and ploidy of blastocysts were evaluated. To this aim, COC were aspirated from slaughtered ovaries and IVM for 22 h. Oocytes were activated with 5 µM of Io for 4 min and then randomly allocated into 1 of the following treatments: 25 or 50 µM ROSC or 10 µg mL–1 of CHX for 5 h; 15 or 30 µM DhL for 3 h; or 5 min of exposure to 7% ethanol 4 h post-Io. Controls were Io followed by (1) 3 h in TCM-199 and 3 h in 1.9 mM 6-DMAP (Io-3h-DMAP) and (2) 3 h of exposure to 1.9 mM 6-DMAP (Io-DMAP). Oocytes were cultured in SOF medium. The PN formation and 2PB extrusion were assessed by 5 µg mL–1 of propidium iodide oocyte staining, 17 h after Io. Cleavage, morulae, and blastocyst stages were evaluated at Days 2, 5, and 8 of in vitro development, respectively. Chromosome number of blastocysts was evaluated by Giemsa staining. Data were analysed by Fisher's test (P &lt; 0.05). Rates of 2PB extrusion were 75, 61.1, 60, 56.3, 54.6, and 42.9% for 15 µM DhL (n = 23), 50 µM ROSC (n = 22), Io-3h-DMAP (n = 9), CHX (n = 17), 25 µM ROSC (n = 22), and ethanol (n = 22), respectively, with no differences between groups. A PN was observed in over 81% of the oocytes activated with ethanol, 25 µM ROSC, CHX, 50 µM ROSC, and 15 µM DhL. Lower percentages of 2PB extrusion and PN formation were observed for 30 µM DhL (n = 22; 6.3 and 0%, respectively). The highest cleavage rates were 83.2% for 25 µM ROSC (n = 185), not differing from 78% in Io-DMAP (n = 159). Cleavage rates for 50 µM ROSC (n = 185), CHX (n = 143), and ethanol (n = 74; 80.5, 80.4 and 67.6%, respectively) were not different from Io-3h-DMAP (n = 78; 71.8%) and Io-DMAP. Cleavage rates for 15 µM DhL (n = 70) and 30 µM DhL (n = 93) were the lowest (48.6 and 25.8%). Blastocyst rates were the highest for CHX and 50 µM ROSC, not differing from Io-3h-DMAP (21.7 and 10.8 v. 18%). Very few or no blastocysts were obtained with ethanol, 25 µM ROSC, 30 µM DhL, and 15 µM DhL (4.1, 3.8, 1.1, and 0%, respectively), although ethanol was not different from Io-3h-DMAP. Chromosome number analysis showed that ethanol (n = 2) and CHX (n = 2) resulted in a higher percentage of haploid embryos (50% each), followed by 50 µM ROSC (n = 8), 25 µM ROSC (n = 3), and Io-3h-DMAP (n = 8; 37.5, 33.3% and 12.5%, respectively), although they were not different. Remaining embryos were diploid, aneuployd, or mixoployd. In conclusion, DhL and ROSC proved to be as effective as CHX or ethanol regarding 2PB extrusion and resulting ploidy, defining features when activating oocytes in ART, suggesting they could be efficiently used in bovine to assist ICSI.

Reactive oxygen species production and redox state in parthenogenetic and sperm-mediated bovine oocyte activation

The knowledge concerning redox and reactive oxygen species (ROS)-mediated regulation of early embryo development is scarce and remains controversial. The aim of this work was to determine ROS production and redox state during early in vitro embryo development in sperm-mediated and parthenogenetic activation of bovine oocytes. Sperm-mediated oocyte activation was carried out in IVF-modified synthetic oviductal fluid (mSOF) with frozen-thawed semen. Parthenogenetic activation was performed in TALP plus ionomycin and then in IVF-mSOF with 6-dimethylaminopurine plus cytochalasin B. Embryos were cultured in IVF-mSOF. ROS and redox state were determined at each 2-h interval (7-24 h from activation) by 2',7'-dichlorodihydrofluorescein diacetate and RedoxSensor Red CC-1 fluorochromes respectively. ROS levels and redox state differed between activated and non-activated oocytes (P<0.05 by ANOVA). In sperm-activated oocytes, an increase was observed between 15 and 19 h (P<0.05). Conversely, in parthenogenetically activated oocytes, we observed a decrease at 9 h (P<0.05). In sperm-activated oocytes, ROS fluctuated throughout the 24 h, presenting peaks around 7, 19, and 24 h (P<0.05), while in parthenogenetic activation, peaks were detected at 7, 11, and 17 h (P<0.05). In the present work, we found clear distinctive metabolic patterns between normal and parthenogenetic zygotes. Oxidative activity and ROS production are an integral part of bovine zygote behavior, and defining a temporal pattern of change may be linked with developmental competence.

Artificial activation of bovine and equine oocytes with cycloheximide, roscovitine, strontium, or 6-dimethylaminopurine in low or high calcium concentrations

Knowledge on parthenogenetic activation of oocytes is important to improve the efficiency of nuclear transfer (NT) and intracytoplasmic sperm injection (ICSI) because artificial activation of oocyte (AOA) is an essential step to achieve embryo production. Although different procedures for AOA have been established, the efficiency of in vitro production of embryos remains low, especially in equines and Bos taurus bovines. In an attempt to improve the techniques of NT and ICSI in bovine and equine species, we tested different combinations of drugs that had different mechanisms of action for the parthenogenetic activation of oocytes in these animals. The oocytes were collected, in vitro matured for 24 to 30 h and activated artificially, in the presence of low or high concentrations of calcium, with combinations of calcium ionophore (ionomycin) with cycloheximide, roscovitine, strontium, or 6-dimethylaminopurine (6-DMAP). For assessment of activation rates, oocytes were stained with Hoechst 33342 and observed under an inverted microscope. We showed that all combinations of drugs were equally efficient in activating bovine oocytes, with the best results obtained when high concentrations of calcium were adopted. For equine oocytes, high concentrations of calcium were not beneficial for the parthenogenetic activation and the combination of ionomycin with either 6-DMAP or roscovitine was effective in inducing artificial activation of oocytes. We believe that our preliminary findings provide some clues for the development of a better AOA protocol to be used with these species.

Disparate consequences of heat stress exposure during meiotic maturation: embryo development after chemical activation vs fertilization of bovine oocytes

Consequences of heat stress exposure during the first 12 h of meiotic maturation differed depending on how and when bovine oocytes were activated. If heat-stressed oocytes underwent IVF at ~24 h, blastocyst development was less than for respective controls and similar to that obtained for nonheat-stressed oocytes undergoing IVF at 30 h (i.e. slightly aged). In contrast, if heat-stressed oocytes underwent chemical activation with ionomycin/6-dimethylaminopurine at 24 h, blastocyst development was not only higher than respective controls, but also equivalent to development obtained after activation of nonheat-stressed oocytes at 30 h. Developmental differences in chemically activated vs IVF-derived embryos were not related to fertilization failure or gross alterations in cytoskeletal components. Rather, ionomycin-induced calcium release and MAP kinase activity were less in heat-stressed oocytes. While underlying mechanisms are multifactorial, ability to obtain equivalent or higher development after parthenogenetic activation demonstrates that oocytes experiencing heat stress during the first 12 h of meiotic maturation have the necessary components to develop to the blastocyst stage, but fail to do so after fertilization.

Evidence for the Involvement of PKC and Actin Filaments in Endoplasmic Reticulum Organization During Bovine Oocyte Activation.

Evidence for the Involvement of PKC and Actin Filaments in Endoplasmic Reticulum Organization During Bovine Oocyte Activation.

326 MICROINJECTIONS OF SMALL INTERFERING RNA AND COMPLEMENTARY RNA TO ELUCIDATE THE INVOLVEMENT OF ENDOGENOUS PHOSPHOLIPASE C ISOFORMS IN BOVINE OOCYTE ACTIVATION DURING FERTILIZATION

Phospholipase C (PLC) isoforms stimulate the hydrolysis of phosphatidyl inositol (4,5)-bisphosphate (PIP2) to produce diacylglycerol (DAG) and 1,4,5 inositol trisphosphate (IP3), with IP3 regulating the release of calcium (Ca2+) from the endoplasmic reticulum. This release of calcium is essential for oocyte activation, and a sperm-specific PLC isoform, PLCγ;, has been proposed as the primary agent that initiates the activation process. However, the oocyte contains many endogenous PLC isoforms (PLC β, γ, and δ) that could also be involved in regulating or initiating these calcium oscillations downstream of other initiating events. In order to better elucidate the involvement of endogenous PLC isoforms as well as the specific role of the sperm-specific form, small interfering RNA (siRNA) directed against the specific bovine PLC isoforms (PLCζ;, PLCγ1, PLCγ2, PLCδ1, PLCδ3, PLCδ4, PLCβ1, PLCβ3) were microinjected into bovine oocytes, and the subsequent effects on PLC mRNA levels and bovine fertilization were evaluated. Real-time PCR (qPCR) was used to quantify the levels of PLC message present in bovine oocytes at the time of injection (15 h post-maturation) and 6, 10, and 14 h post-injection. The qPCR results indicated a near-complete knockdown of mRNA levels in bovine oocytes 10 h post-injection for the isotypes PLCγ1, PLCγ2, PLCδ3, PLCδ4, PLCβ1, PLCβ3, but only partial knockdown of PLCS 1 mRNA. Oocytes microinjected with PLC siRNA were also fertilized and cultured in vitro according to our standard laboratory procedures (Reed et al. 1996 Theriogenology 45, 439-449). The oocytes microinjected with PLCζ;, PLCδ1, PLCδ3, PLCδ4, PLCβ1, PLCβ3 siRNA resulted in cleavage rates similar to the negative control siRNA, non-injected, and sham-injected treatment groups, whereas bovine oocytes microinjected with PLCγ1 and PLCγ2 siRNA had significantly lower cleavage rates compared with the controls. Additionally, complementary cRNA for each specific PLC isoform was microinjected into bovine oocytes to ascertain each isoform’s ability to induce parthenogenetic activation. Development was observed in oocytes microinjected with a variety of cRNAs, and the activating effects of the cRNA were negligible if the oocytes were microinjected with the corresponding siRNA before microinjection with cRNA. Interestingly, siRNA specific for PLCζ; failed to reduce cleavage when treated bovine oocytes were fertilized. These data illustrate the potential involvement of multiple endogenous PLC isoforms and not just the sperm-specific PLCζ; isoform in bovine oocyte activation during fertilization.

A combined treatment of ionomycin with ethanol improves blastocyst development of bovine oocytes harvested from stored ovaries and microinjected with spermatozoa

Regardless of the presence of sperm-borne oocyte-activating factors, activation of bovine oocytes with exogenous activation stimuli is required for further development after intracytoplasmic sperm injection (ICSI). The current study was designed to develop a new activation regimen for improving the blastocyst yield after ICSI of bovine oocytes harvested from ovaries stored at 10 to 12 °C for 24 h. After ICSI, oocytes were treated with 5 μM ionomycin for 5 min, 7% ethanol for 5 or 10 min, ionomycin followed by ethanol (5 or 10 min), ionomycin followed by 10 μg/mL cycloheximide for 5 h, or ionomycin followed by 1.9 mM 6-dimethylaminopurine for 3 h. Across the activation regimens, the cleavage rates of ICSI oocytes (45% to 77%) were higher than those of parthenogenetically activated oocytes (11% to 21%; P < 0.05). Activating the ICSI oocytes with ionomycin plus ethanol improved the blastocyst yield (29% to 30%) compared with that of nontreated oocytes (12%; P < 0.05), but the other regimens did not improve the blastocyst yield (9% to 18%; P > 0.05). Higher blastocyst yields were due to increasing the proportion of ICSI oocytes that passed through the early postfertilization events until cleavage. None of the regimens have any adverse effect on the quality of the blastocysts regarding the total cell number or the proportion of the inner cell mass cells. Thus, a new activation regimen using two triggers for single calcium increase effectively improved blastocyst yield after bovine ICSI using oocytes harvested from stored ovaries.