Mos–MAPK inhibition prevents MII leading to parthenogenetic development. (A) Western blot of a time course using phospho-MAPK (marking activated MAPK) and phospho-Cdk1 (marking the inactive Tyr15 phosphorylated form) antibodies to monitor Mos–MAPK and Cdk1 activities during meiosis of starfish oocytes. Oocytes were treated with 20 µM U0126 or an equal amount of DMSO added simultaneously with the maturation hormone. Time points are indicated as minutes after NEBD. Fertilization was performed 60 min after NEBD. Numbers to the left are in kDa and mark the position of the molecular weight markers. For the complete blots see Source Data F1. (B) Selected frames from time-lapse recording of oocytes expressing H2B-mCherry (red) and injected with HiLyte488-tubulin (cyan) to label chromosomes and microtubules, respectively. Oocytes were imaged through meiosis and then fertilized after MI while imaging on a spinning disk confocal microscope. Before inducing maturation, oocytes were treated either with 20 µM U0126 or an equal amount of DMSO. PBI and outlines indicate the polar bodies. Venus and Mars signs indicate female and male pronuclei, respectively. Insets show magnified views of chromosomes. Maximum intensity projections are shown, scale bars are 20 µm (insets 5 µm), and time is given in minutes. (C) Individual data points and a boxplot showing the time from NEBD to metaphase I quantified on recordings similar to those shown in B. Statistical comparison was done using the Mann–Whitney test; n marks the number of oocytes imaged. (D) Individual data points and a boxplot showing the time from meiotic metaphase I to the first mitotic metaphase quantified on recordings similar to those shown in B. Statistical comparison was done using the Mann–Whitney test, n marks the number of oocytes imaged. (E) Immunofluorescence images of oocytes stained for microtubules (anti-tubulin, cyan), chromatin (Hoechst 33342, red), and the lamina (anti-lamin B, green). Oocytes were treated with 20 µM U0126 or an equal amount of DMSO and then fixed 60 and 120 min after NEBD, respectively. Samples were imaged on a spinning disk microscope. Maximum intensity projections are shown; scale bars are 10 µm. (F) Selected frames from a time-lapse recording of oocytes expressing H2B-mCherry (red) and injected with HiLyte488-tubulin (cyan) to label chromosomes and microtubules, respectively. Oocytes were imaged through meiosis on a spinning disk confocal microscope. Before inducing maturation, oocytes were treated with 20 µM U0126. Then oocytes were either left unmanipulated, or they were injected with active Cdk1–cyclin B protein immediately following extrusion of the first polar body. Maximum intensity projections are shown, scale bars are 20 µm, and time is given in minutes. PB I denotes the first polar body, and PN is the pronucleus. Source data are available for this figure: SourceData F1.
Figure 1.

Mos–MAPK inhibition prevents MII leading to parthenogenetic development. (A) Western blot of a time course using phospho-MAPK (marking activated MAPK) and phospho-Cdk1 (marking the inactive Tyr15 phosphorylated form) antibodies to monitor Mos–MAPK and Cdk1 activities during meiosis of starfish oocytes. Oocytes were treated with 20 µM U0126 or an equal amount of DMSO added simultaneously with the maturation hormone. Time points are indicated as minutes after NEBD. Fertilization was performed 60 min after NEBD. Numbers to the left are in kDa and mark the position of the molecular weight markers. For the complete blots see Source Data F1. (B) Selected frames from time-lapse recording of oocytes expressing H2B-mCherry (red) and injected with HiLyte488-tubulin (cyan) to label chromosomes and microtubules, respectively. Oocytes were imaged through meiosis and then fertilized after MI while imaging on a spinning disk confocal microscope. Before inducing maturation, oocytes were treated either with 20 µM U0126 or an equal amount of DMSO. PBI and outlines indicate the polar bodies. Venus and Mars signs indicate female and male pronuclei, respectively. Insets show magnified views of chromosomes. Maximum intensity projections are shown, scale bars are 20 µm (insets 5 µm), and time is given in minutes. (C) Individual data points and a boxplot showing the time from NEBD to metaphase I quantified on recordings similar to those shown in B. Statistical comparison was done using the Mann–Whitney test; n marks the number of oocytes imaged. (D) Individual data points and a boxplot showing the time from meiotic metaphase I to the first mitotic metaphase quantified on recordings similar to those shown in B. Statistical comparison was done using the Mann–Whitney test, n marks the number of oocytes imaged. (E) Immunofluorescence images of oocytes stained for microtubules (anti-tubulin, cyan), chromatin (Hoechst 33342, red), and the lamina (anti-lamin B, green). Oocytes were treated with 20 µM U0126 or an equal amount of DMSO and then fixed 60 and 120 min after NEBD, respectively. Samples were imaged on a spinning disk microscope. Maximum intensity projections are shown; scale bars are 10 µm. (F) Selected frames from a time-lapse recording of oocytes expressing H2B-mCherry (red) and injected with HiLyte488-tubulin (cyan) to label chromosomes and microtubules, respectively. Oocytes were imaged through meiosis on a spinning disk confocal microscope. Before inducing maturation, oocytes were treated with 20 µM U0126. Then oocytes were either left unmanipulated, or they were injected with active Cdk1–cyclin B protein immediately following extrusion of the first polar body. Maximum intensity projections are shown, scale bars are 20 µm, and time is given in minutes. PB I denotes the first polar body, and PN is the pronucleus. Source data are available for this figure: SourceData F1.

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