In vitro development of spontaneously activated bovine oocytes

Despite enormous advances in the identification and sequencing of developmentally relevant bovine genes, the function of the majority of these transcripts is not yet known. Here we aimed to apply the RNA interference (RNAi) approach to suppress the expression of the maternal transcript c-mos (AY630920) and embryonic transcripts E-cadherin (AY508164) and Oct-4 (AY490804) during in vitro development of bovine embryos using microinjection of sequence-specific double-stranded RNA (dsRNA). For this 435-, 341- and 341-bp-long dsRNA specific to the coding sequences of c-mos, E-cadherin and Oct-4 transcripts, respectively, were synthesized using Promega RiboMax" T7 system (Promega, Madison, WI, USA), where sense and antisense strands were transcribed from the target DNA template. Slaughterhouse ovaries were used to aspirate bovine oocytes, which were matured in TCM-199 with 12% estrus cow serum (ECS), fertilized in Fert-TALP, and cultured in CR1 medium at 39�C under humidified atmosphere of 5% CO2 in air. In Experiment 1, immature oocytes were categorized into three groups, each containing 50-60 oocytes: those injected with c-mos dsRNA, those injected with RNase-free water, and uninjected controls. In Experiment 2, zygotes were categorized into four groups, each containing 50-60 zygotes: those injected with E-cadherin dsRNA, those injected with Oct-4 dsRNA, those injected with RNase-free water, and uninjected controls. Each experiment was repeated four times. The effect of dsRNA on in vitro development of oocytes or embryos was assessed after microinjection during culture. The level of mRNA and protein expression was investigated using real-time PCR and western blot analysis, respectively. Data were analyzed using SAS, version 8 (SAS Institute Inc., Cary, NC, USA). Microinjection of c-mos dsRNA resulted in a 70% reduction of c-mos transcript abundance after maturation compared to the water-injected and uninjected controls (P < 0.05). Similarly, microinjection of E-cadherin and Oct-4 dsRNA at the zygote stage resulted in 80% and 60% reduction in transcript abundance at the blastocyst stage, respectively, compared to the uninjected controls (P < 0.05). Decreases in the c-mos (39 kDa) and E-cadherin proteins (119 kDa) were observed in the c-mos and E-cadherin dsRNA-injected groups, respectively, compared to the control. A higher proportion of oocytes (75%) showed first polar body extrusion after maturation in c-mos dsRNA-injected groups, compared to 52% in water-injected and 57% in uninjected controls. Only 22% from E-cadherin dsRNA- and 24% from Oct-4 dsRNA-injected zygotes developed to the blastocyst stage compared to 39 and 37% blastocyst rates in water-injected and uninjected control groups, respectively. In conclusion, injection of sequence-specific dsRNA in bovine oocytes and embryos resulted in suppression of mRNA and their protein products, thereby affecting in vitro development of bovine embryos.

Download Full-text

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

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab273 ◽

2015 ◽

Vol 27 (1) ◽

pp. 225

Author(s):

M. Suvá ◽

N. G. Canel ◽

D. F. Salamone

Keyword(s):

Chromosome Number ◽

Polar Body ◽

Routine Procedure ◽

In Vitro Development ◽

Second Polar Body ◽

N 93 ◽

Haploid Embryos ◽

Vitro Development ◽

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 < 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.

Download Full-text