Nk2 micromanipulator

Embryos were dechorionated and glued to a coverslip as above, dehydrated for 10–15 min, and covered with a 1:1 mix of halocarbon oil 27 and 700 (Sigma-Aldrich). Embryos were injected using a Transferman NK2 micromanipulator (Eppendorf, Hamburg, Germany), and a PV820 microinjector (WPI, Sarasota, FL) attached to the spinning disk confocal microscope. Drugs (Y-27632, Tocris Bioscience, Bristol, UK); (Cytochalasin D, EMD Millipore, Darmstadt, Germany) were injected into the perivitelline space, where they are predicted to be diluted 50-fold (Foe and Alberts, 1983 (link)). Y-27632 was injected at 100 mM in water; control embryos were injected with water. Cytochalasin D was injected at 5 mM in 50% DMSO; control embryos were injected with 50% DMSO. Embryos were imaged immediately after injection for at least 10 min.

A droplet of 5 uL of the mitochondrial suspension was placed in a microinjection dish next to the sperm, wash and oocyte droplets. All droplets were covered by light mineral oil (Sigma, Sydney, Australia). ICSI and mitochondrial supplementation were performed under an inverted microscope (Olympus IX71, Tokyo, Japan) equipped with an attached NK2 micromanipulator (Eppendorf AG, Eppendorf, Germany). ICSI was performed by catching a single sperm cell using a microinjection pipette (The Pipette Company, Origio, Adelaide, Australia), then injected into an oocyte that was held by a holding pipette (The Pipette Company), whilst mitochondrial supplementation was performed by catching a single sperm and then expelling it into the drop containing the mitochondria. The sperm cell was recaptured again and aspirated into the injection pipette along with ~3pl mitochondrial isolate. The sperm cell and mitochondria were then injected into the oocyte. Only surviving and non-lysed oocytes progressed to in vitro embryo culture.

The overexpression of miR-145 and miR-145-mu were attained through purchasing miR-145 mimic\mu from GenePharma and diluting them to a final concentration of 20 uM. To overexpress Abca1, a capped cRNA was synthesized as described below. An Eppendorf Transferman NK2 micromanipulator was utilized to conduct the microinjection of the miRNA mimic\mu,cRNA or distilled water (Control), to denuded oocytes with an injection volume of 5-10 pL. In addition, a group of embryos that had not undergone microinjection served as culture controls (Normal). Once this injection was complete, oocytes were rinsed and cultured in an M16 medium for 14 h.