The effect of Duration of Sheep Oocyte In Vitro Maturation on Induction of Parthenogenesis Using Ionomycin and 6-DMAP

Bahiraee A *, Shirazi A , Ahmadi E , Nazari H , Ahmadi E , Nazari H , Borjian S ,


Objective: The aim of this study was to compare the effect of time of partenogenetic activation (22 h versus 27 h after IVM) on subsequent development of IVM-derived ovine oocytes using either single activation agents (ionomycin 5 μM for 5 min) or combined activation treatments (ionomycin with 6-DMAP 2.0 mM for 3 h). Cumulus oocyte complexes (COCs) were recovered from abattoir-derived ovaries and matured in vitro. After maturation cumulus-free oocytes were activated according to the experiment designs. Activated oocytes were cultured in vitro in modified synthetic oviductal fluid medium and assessed for the cleavage blastocyst and hatching rates. Compared with IVF controls the oocytes activated after 22 h of IVM using only ionomycin had a reduction in cleavage blastocyst and hatching rates whereas the addition of the 6-DMAP an MPF-inhibitor especially to oocytes activated after 27 h of IVM restored development measured by cleavage and hatching rates. However the addition of 6-DMAP did not cause any positive effect on cell density/allocation between parthenote groups. Nonetheless irrespective of the activation protocol development to the blastocyst stage total cell number and cell allocation (ICM/total cells) were significantly lower in parthenogenetic embryos than controls. In conclusion compared with 22 h of IVM it seems that a prolonged IVM time (27 h) and the addition of 6-DMAP improve embryo developmental potential of ovine parthenogenetic embryos. Materials and Methods: Materials and methods Except where otherwise indicated all chemicals were obtained from the Sigma (St. Louis MO USA). Oocyte collection and in vitro maturation The method for in vitro production of sheep embryo with slightly modification was the method of Thompson et al (1995). Oocytes used in this study were recovered from prepubertal and adult ovine ovaries collected at a local slaughterhouse and transported to the laboratory within 2 to 3 h in normal saline at temperature between 25 and 35 °C. Ovaries were washed 3 times with prewarmed fresh saline (37C) and all visible follicles with a diameter of 2 to 6 mm were aspirated using gentle vacuum (30 mm Hg) via a 20 gauge short beveled needle connected to a vacuum pump. The follicle content released in preincubated hepes-modified TCM supplemented with 50 IU/ml heparin. The cumulus–oocyte complexes (COCs) with at least 3 layers of cumulus cells oocytes with a uniform granulated cytoplasm homogenous distribution of lipid <font>drop</font>lets in the cytoplasm were selected for the experiments. Before culturing oocytes were washed in Hepes-buffered TCM199 (H-TCM199) supplemented with 5% FBS (Fetal bovine serum Gibco 10270) and 2mM glutamine. The oocyte culture medium (OCM) consisted of bicarbonate-buffered TCM 199 with 2 mM L-glutamine supplemented with 0.02 mg/ml cysteamine 1 IU/ml hCG 1 µg/ml E2 100 µl/ml penicillin 100 µg/ml streptomycin 10% FBS (Fetal bovine serum Gibco 10270) and 0.2 mM Na-Pyrovate. The COCs were randomly distributed in maturation <font>drop</font>lets (10 oocytes in 50 µl) and covered by sterile paraffin oil in a 60-mm Petri dish (Falcon 1008; Becton & Dickinson Lincoln Park NJ) and were then incubated under an atmosphere of 5%CO2 -95% air with 100% humidity at 39 ?C for 22 and 27 h. Preparation of sperm and in vitro fertilization The matured COCs were washed four times in H-SOF (HEPES- Synthetic Oviduct Fluid) solution and once in fertilization medium (SOF enriched with 20% heated inactivated estrous sheep serum) and then placed in 45 ?l <font>drop</font>lets (10 COCs per <font>drop</font>let) of fertilization medium. Fresh semen was collected from a Lori-Bakhtiari breed ram of proven fertility. For swim up 80-100 ?l of semen was kept under 1 ml of BSA-HSOF in 15 ml conical Falcon tube at 39C for up to 45 min. After swim up the 700-800 l of the top fluid were then added to 3 ml of BSA-HSOF centrifuged twice at 200 × g for 3 min and the final pellet was resuspended with BSA-HSOF. A 5 l aliquot of sperm suspension were added to the fertilization <font>drop</font>s at a concentration of 1 × 106 sperm/ml Incubation was carried out at 39 °C in 5% CO2 in air with saturated humidity for 22 h. Twenty two hours after insemination presumptive zygotes were denuded of surrounding cumulus cells by vortexing in H-FOS and transferred to culture <font>drop</font>lets. Activation of oocyte Methods for activation of oocytes were modified from Susko-Parrish et al. (1994). After IVM (22 h and 27 h) cumulus cells were removed by incubation in 0.1% hyaluronidase in H-SOF at 38.5 °C for 2 min followed by vortexing for 3 min. Denuded oocytes were randomly allocated into single or combined treatment groups. Oocytes were treated with single activation agents including ionomycin (5 M for 5 min. After 5 min exposure to ionomycin the oocytes were then rinsed in H-SOF containing 30mg/ml BSA to stop activation. All of the chemicals for oocyte activation were dissolved in H-SOF medium supplemented with 1 mg/ml bovine serum albumin (BSA). For the combined treatment oocytes were firstly activated with the same concentrations of ionomycin as in the single treatment and then were immediately incubated in 2.0 mM 6-DMAP for extra 3 h. Following activation oocytes were washed twice with the H-SOF medium and transferred to the culture medium. In vitro culture Presumptive zygotes in IVF groups and activated oocytes in parthenogenetic groups were allocated to 20 l culture <font>drop</font>s (five to six embryos/<font>drop</font>) consisting of SOF supplemented with 2% (v/v) BME-essential amino acids 1% (v/v) MEM-nonessential amino acids 1mM glutamine and 8 mg/ml fatty acid free BSA. Embryo culture took place under mineral oil in a humidified atmosphere of 5% CO2 7% O2 at 39 C. On the third and fifth day of culture (Day 0 defined as the day of fertilization) 10% charcoal stripped fetal bovine serum (FBS) was added to the medium. The percentage of cleaved embryos at day 3 and the percentage of blastocysts at day 7 were expressed on the basis of the number of oocytes at the onset of culture and the percentage of hatched blastocysts at day 8 expressed on the basis of the total number of blastocysts present at day 7. The summary of experimental groups is presented in the below. Each experiment was consisted of at least 5 replicates. Group IVF: The in vitro matured oocytes for 22 h and 27 h were fertilized with fresh semen as control. Group Io: Denuded oocytes activated with 5 µm Ionomycin for 5 min. Group Io + 6-DMAP: Denuded oocytes activated with 5 µm Ionomycin for 5 min + 2 mM 6-DMAP for 3 h Differential staining Differential staining of inner cell mass (ICM) and trophectoderm (TE) compartments was carried out on day 7 blastocysts. Briefly blastocysts were incubated in Triton X-100 prepared in the base medium (H-SOF containing 5 mg BSA/ml) for 20 seconds. The blastocysts were then stained in the base medium containing 30 µg/ml propidium iodide (PI) for 1 min. After two washes in the base medium the blastocysts were transferred in ice-cold ethanol containing 10 mg/ml Hoechst 33342 for 15 min. The blastocysts were directly mounted into the small <font>drop</font>let of glycerol on glass slide and examined under an epifluorescent microscope (IX71 Olympus Tokyo Japan). ICM nuclei appeared blue caused by DNA labeling with the membrane permeable Hoechst 33342 and trophoblastic cells appeared red due to staining of nuclear DNA with the membrane impermeable PI. Results: Results Effect of oocyte age on effectiveness of parthenogenetic treatments on the development of parthenotes As shown in Table 1 two maturation times were considered to compare the effect of four activation regimens on parthenogenesis of ovine oocytes matured in vitro. The cleavage rates of artificially activated oocytes after 22h of culture in group receiving either Io was lower than groups receiving Io + 6-DMAP. The cleavage rate in combined treatment groups (Io + 6-DMAP) was comparable with IVF group. There was no significant difference between IVF and oocytes artificially activated after 27h of culture in term of cleavage rate. The cleavage rates were significantly increased in groups Io when the maturation time was increased from 22h to 27h. The balstocyst rates of artificially activated oocytes after 22h of culture in group receiving either Io was lower than groups receiving Io + 6-DMAP. The blastocyst rates in groups receiving Io + 6-DMAP after 27h of culture was higher than groups receiving either Io. The corresponding rate in IVF group however was significantly higher than parthenogenetically activated oocytes at both 22h and 27h of culture. The blastocyst rates likewise cleavage rates were significantly increased in groups Io when the maturation time was increased from 22h to 27h. The hatching rates in combined treatment groups after both 22h and 27h of culture were significantly higher than single treatment group. The corresponding rate however was significantly higher in IVF group compared to artificially activated oocytes after 22h of culture. The hatching rate was significantly increased in group Io + 6-DMAPas the maturation time was increased to 27h. Conclusion: it seems that compared with 22 h of IVM a prolonged IVM time (27 h) and the addition of 6-DMAP improve embryo developmental potential of ovine parthenogenetic embryos