1 methyladenine 1 ma

Starfish (Patiria miniata or P. pectinifera) were obtained in the springtime from Southern California (South Coast Bio-Marine LLC, Monterey Abalone Company or Marinus Scientific Inc) or were kindly provided by Kazoyushi Chiba (Ochanomizu University, Tokyo, Japan). They were kept at 16°C for the rest of the year in seawater aquariums at EMBL’s or MPI-BPC’s marine facilities. Oocytes were extracted from the animals fresh for each experiment as described earlier (Lénárt et al., 2003 (link)). mRNAs and other fluorescent markers were injected using microneedles, as described previously (Jaffe and Terasaki, 2004 (link); Borrego-Pinto et al., 2016 (link)). mRNA was injected the day before to allow protein expression, whereas fluorescently labeled protein markers or dextrans were injected a few hours prior to imaging. Meiosis was induced at the initiation of the experiment by addition of 1-methyladenine (1-MA, 10 µM, Acros Organics). NEBD normally started 20–25 min after 1-MA addition, and only oocytes that initiated NEBD within 40 min of 1-MA addition were considered. Every experiment was repeated at least three times, with oocytes taken from at least two different animals.

Astropecten aranciacus (starfish) were collected from the end of January to May in the Gulf of Naples and Gaeta and maintained at 16 °C in circulating seawater. GV-stage oocytes were isolated from the ovary by making a small hole in the dorsal region of the female animal. The oocytes were sieved with gauze and collected in natural seawater (NSW, pH 8.1) filtered with a Millipore membrane of 0.2 µm pore size (Nalgene vacuum filtration system, Rochester, NY, USA). The collected oocytes kept in NSW were used for maturation and fertilization experiments within the next 3 hr. The dry sperm collected from the testis were diluted in NSW and used to inseminate oocytes and eggs at a final concentration of 1 × 10⁶ sperm/mL. In vitro maturation was performed by adding the hormone 1-methyladenine (1-MA) (Acros Organics, Fisher Scientific, Milan, Italy) to the GV-stage oocytes suspended in NSW at different pH at a final concentration of 10 μL/mL. The lowering of pH was obtained immediately before the experiment by adding HCl to NSW (pH 8.1) until reaching the needed value (pH 6.8) [39 (link)]. The pH of seawater was raised to 9.0 by adding sufficient NH₄OH to NSW (pH 8.1), about 1 mmol of NH₄OH/l, which was previously used in studying membrane potential and cortical F-actin structural changes upon the exposure of sea urchin eggs to NH₄OH seawater [40 (link),44 (link)].

Starfish (Patiria miniata) were obtained from Southern California (South Coast Bio-Marine LLC, Monterey Abalone Company, or Marinus Scientific Inc). Animals were maintained in seawater aquariums at 16°C at European Molecular Biology Laboratory’s Marine Facility. Oocytes were isolated, and mRNAs and other fluorescent markers were injected into the oocytes by using mercury-filled microneedles, as described previously (Jaffe and Terasaki, 2004 (link); Borrego-Pinto et al., 2016b (link)). mRNA was injected 24–48 h before to allow protein expression, whereas fluorescently labeled protein markers were injected a few hours before imaging. Oocytes were induced to enter meiosis by addition of 10 µM 1-methyladenine (1-MA; Acros Organics). NEBD normally initiated 25 min after 1-MA addition, and only those oocytes that started NEBD within 40 min were considered for analysis.