Effects of Activation Methods on DNA Synthesis and Development of Parthenogenetic Porcine Embryos

Abstract

This study investigated the timing of DNA synthesis and patterns of pronuclear (PN) formation during the first cell cycle, and its influence on developmental competence, velocity and proliferation index of porcine parthenote blastocysts produced by different activation treatments. Oocytes were activated as follows: electrical stimulation (EST), EST combined with 7.5 μg/ml cytochalasin B (EST + CCB), 10 μg/ml cycloheximide (EST + CHX) and 1.9 mm 6-dimethylaminopurine (EST + 6-DMAP) for 3 h. DNA synthesis and PN formation were evaluated using 1 mm 5'bromo-2'deoxy-uridne (BrdU) at 2 h intervals from 1 to 13 h or 5 to 13 h of post-activation (hpa), respectively. In EST, DNA synthesis started at 3 hpa, reached the peak at 11 hpa and decreased at 13 hpa. Treatment with 6-DMAP resulted in an early increase of DNA synthesis at 3 hpa, whereas CCB delayed DNA synthesis for 2 h. In EST and EST + 6-DMAP, most of the eggs showed 1PN, whereas, incidence of 2PN in EST + CCB was higher than 1PN. EST + CHX was observed with 1PN, 2PN and multiple PN. Blastocyst rate in EST + CCB and EST + 6-DMAP were significantly (p<0.05) higher than EST + CHX. But, the developmental velocity was not different among groups. Proliferation index of blastocysts, as indicated the number of blastomere at S-phase of the cell cycle was low in all groups. In conclusion, CCB, CHX and 6-DMAP used for producing porcine parthenogenetic embryos induced different onset of DNA synthesis and PN, but they did not affect the subsequent embryo development.

Comparison of chemical, electrical, and combined activation methods for in vitro matured porcine oocytes

“…We found that combined activation significantly improved the development potential of parthenogenetic embryos than chemical activation or electrical activation only. It was consistent with the previous studies in cattle (Hosseini et al 2008), goat (Kumar et al 2014), and pig (Ock et al 2011). Comparing with Ion chemical treatment, combined treatment of electrical stimuli induced more Ca 2+ oscillations, and it could mimic intracellular events induced by sperm penetration more efficiently (Sun et al 1992).…”

Development and Gene Expression of Porcine Cloned Embryos Derived from Bone Marrow Stem Cells with Overexpressing Oct4 and Sox2

“…Bone marrow-derived MSCs (BMSCs) from different sources and passages revealed different characteristics regarding differentiation ability, fibroblast-like morphology, proliferation ability, and expression of transcription factors. Oct4, Sox2, and Nanog, which are known as important early transcription factors for regulation of stem cells pluripotency, are expressed in MSCs (Do and Schöler, 2009;Ock et al, 2011). Deletion of Oct4, Sox2, and Nanog in embryonic stem cells (ESCs) resulted in loss of pluripotency and failure to maintain the epiblast (Welstead et al, 2008).…”

Wedelolactone facilitates the early development of parthenogenetically activated porcine embryos by reducing oxidative stress and inhibiting autophagy

“…In brief, after IVM, oocytes and cumulus cells were separated from COCs by pipetting approximately 30 times with 0.1% hyaluronidase in a 1.5 mL centrifuge tube ( Van de Velde et al, 1997 ). The cumulus-depleted oocytes were subjected to parthenogenetic activation using two pulses of direct current at 120 V for 60 µs in 300 mM mannitol containing 0.5 mM HEPES, 0.05 mM CaCl 2 •2H 2 O, 0.1 mM MgSO 4 •7H 2 O, and 0.01% polyvinyl alcohol (PVA) and cultured in 7.5 mg/mL cytochalasin B at 38.5 °C in an atmosphere of 5% CO 2 and 100% RH for 3 h ( Ock et al, 2011 ). Subsequently, approximately 40 oocytes were washed thoroughly with the IVC medium, transferred to 500 mL of IVC medium (bicarbonate-buffered PZM-5 supplemented with 4 mg/mL of bovine serum albumin (BSA)) covered with mineral oil in a 4-well culture plate, and incubated at 38.5 °C in an atmosphere of 5% CO 2 and 100% RH for 7 days.…”