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Spire-Type Actin Nucleators Cooperate with Formin-2 to Drive Asymmetric Oocyte Division

Summary Oocytes mature into eggs by extruding half of their chromosomes in a small cell termed the polar body. Asymmetric oocyte division is essential for fertility [1], but despite its importance, little is known about its mechanism. In mammals, the meiotic spindle initially forms close to the center of the oocyte. Thus, two steps are required for asymmetric meiotic division: first, asymmetric spindle positioning and second, polar body extrusion. Here, we identify Spire1 and Spire2 as new key factors in asymmetric division of mouse oocytes. Spire proteins are novel types of actin nucleators that drive nucleation of actin filaments with their four WH2 actin-binding domains [2–6]. We show that Spire1 and Spire2 first mediate asymmetric spindle positioning by assembling an actin network that serves as a substrate for spindle movement. Second, they drive polar body extrusion by promoting assembly of the cleavage furrow. Our data suggest that Spire1 and Spire2 cooperate with Formin-2 (Fmn2) to nucleate actin filaments in mouse oocytes and that both types of nucleators act as a functional unit. This study not only reveals how Spire1 and Spire2 drive two critical steps of asymmetric oocyte division, but it also uncovers the first physiological function of Spire-type actin nucleators in vertebrates.

(J) The mean intensity of the cytoplasmic phalloidin staining was measured in oocytes as shown in (I) that were injected with control siRNA (Control; black), with Spire1 siRNAs (Spire1 RNAi; red) and Spire2 siRNAs (Spire2 RNAi; red). Data are mean±s.d.. P-values were calculated with Student's t-test.
(D) Polar body extrusion efficiency was scored for oocytes that progressed into anaphase and were injected with scrambled negative control siRNA (Control), Spire1+2 siRNAs (Spire1+2 RNAi), Spire1+2 siRNAs together with human Spire1 (Rescue (Spire1)) or human Spire2 mRNAs (Rescue (Spire2)). Oocyte numbers are indicated in white on the columns. (B) The mean intensity of the cytoplasmic phalloidin staining was measured in oocytes as shown in (A) that were injected with 10 pl of 60 µg/ml BSA, 60 µg/ml Exoenzyme C3 (final c in oocyte: 120 nM), exposed to 3 µg/ml Toxin B or injected with mRNA encoding Rac1T17N or Cdc42T17N. Data are mean±s.d.. P-values were calculated with Student's t-test.
Quantification of the density of the actin network revealed that Rho GTPases are not required to activate Spire1/Spire2 and Fmn2 dependent actin nucleation in mouse oocytes.
Instead, inhibition of Cdc42 and Rac1 and treatment with Toxin B slightly increased the density of the actin network. This could be due to a direct inhibitory effect of Rho GTPases 10 on Spire1/Spire2 and Fmn2 dependent F-actin nucleation, or due to indirect effects because more monomeric actin is available for Spire1/Spire2 and Fmn2 when other actin nucleators that are Cdc42 and Rac1 dependent are inactive.
(D) The mean cytoplasmic phalloidin staining was measured in oocytes as shown in (C) that were injected with different concentrations of mRNAs encoding Spire1-mCherry, Spire2-mCherry and Fmn2-mCherry (red columns; 1x, 3x and 10x) and compared to the intensity in wildtype oocytes (black columns; 1x, 3x and 10x). Data are means s.d. P-values were calculated with Student's t-test.

Measurement of the cytoplasmic network density
The quantify the density of the cytoplasmic actin network, we measured the mean intensity of Alexa Fluor 488 phalloidin staining in the cytoplasm and in a region outside of the oocyte for background subtraction. Images in control and perturbed situations were acquired with identical imaging conditions and care was taken that images were not saturated during acquisition. To accurately measure comparable intensities in different oocytes, images for quantification were always acquired in the centre of the oocyte as determined by the maximum radius of the oocyte. Average, standard deviation, and statistical significance based on Student's t-test were calculated in Excel.

Automated 3D tracking of the spindle
To measure the kinetics of asymmetric spindle positioning, we injected oocytes with mRNA encoding fluorescently labelled -tubulin or MAP4 to label microtubules. We then recorded z-stacks of the entire oocyte volume during asymmetric spindle positioning using Zeiss' MultiTime Series macro. We corrected for drifts during image acquisition with the 'correct drift' function of Imaris (Bitplane) after segmenting the oocyte volume by low thresholding on the soluble pool of the fluorescent reporter.
Afterwards we segmented the spindle by applying a higher threshold and tracked the spindle's centre of mass during asymmetric positioning using Imaris.
For averaging of asymmetric spindle positioning in different oocytes, we temporally aligned the different data sets to the time when the spindle slowed down due to arrival at the cortex. In Spire1 and Spire2 co-depleted oocytes or oocytes from