Nampt-mediated spindle sizing secures a post-anaphase increase in spindle speed required for extreme asymmetry

Meiotic divisions in oocytes are extremely asymmetric and require pre- and post-anaphase-onset phases of spindle migration. The latter induces membrane protrusion that is moulded around the spindle thereby reducing cytoplasmic loss. Here, we find that depleting the NAD biosynthetic enzyme, nicotinamide phosphoribosyl-transferase (Nampt), in mouse oocytes results in markedly longer spindles and compromises asymmetry. By analysing spindle speed in live oocytes, we identify a striking and transient acceleration after anaphase-onset that is severely blunted following Nampt-depletion. Slow-moving midzones of elongated spindles induce cortical furrowing deep within the oocyte before protrusions can form, altogether resulting in larger oocyte fragments being cleaved off. Additionally, we find that Nampt-depletion lowers NAD and ATP levels and that reducing NAD using small molecule Nampt inhibitors also compromises asymmetry. These data show that rapid midzone displacement is critical for extreme asymmetry by delaying furrowing to enable protrusions to form and link metabolic status to asymmetric division.


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Life sciences study design
All studies must disclose on these points even when the disclosure is negative. . For immunofluorescence studies, we ensured that oocytes at very similar maturation stages were used by restricting analyses to oocytes that underwent GVBD within 0.5 h of release from IBMX. Usually about 11% of oocytes undergo GVBD within 0.5 h. For immunoblotting, we didn't predetermine the sample size. We tested each antibody to determine how many oocytes (using a minimum of 15 oocytes) were required to produce a clear band whose intensity could be reproducibly quantified. All experiments were performed at least 3 times and involved oocytes obtained from at least 3 mice.
Throughout the paper, oocytes from an inbred BL6/CBAF1 strain were used. Females at 3-4 weeks were selected at random to be hormonally primed prior to being euthanased for obtaining oocytes. Only fully-grown cumulus-covered oocytes that underwent GVBD within 2 h of release from IBMX were included in experiments as is the standard for research with mouse oocytes. For experiments requiring measurements for bipolar spindles, we only included oocytes whose spindles remained in the same horizontal plane throughout the postanaphase-onset period of migration. For experiments requiring detailed measurements for the spindles, protrusions and bulges at perianaphase, we only included oocytes whose spindles remained in the same horizontal plane, the same orientation and could be visualised concurrently with the emerging protrusion (membrane or cortex). This was important because any deviation of the spindle from the horizontal would impact the measurement value.
All experiments were performed at least 3 times and a minimum of 10 oocytes were used for the measurements. Usually variance among the samples was not big as the dots of the samples were clustered in the graph. Importantly, the phenotypes caused by Nampt-depletion were consistent and showed statistically significant differences from controls. We do note that in one case, the variability within the Namptdepletion group was larger than the control group. In contrast to controls in which furrowing occurred roughly halfway along the spindle, following Nampt-depletion, membrane ingression occurred more randomly along the spindle length, typically in an off-centre position closer to the leading spindle pole (Fig. 5d). That's why the variability within the Nampt-depletion group was larger and it was one of our discoveries in the manuscript. Although the variability within the Nampt-depletion group was larger, the phenotype was very reproducible. So all attempts at replication were successful.
For a typical experiment, 3 or more mice were hormonally primed~44-46 h prior to euthanasia for obtaining ovaries. Oocytes from all ovaries were then pooled together and randomly allocated to different treatment groups.
The investigators were not blinded. Using oocytes of differing growth sizes or that undergo GVBD at widely differing times could introduce an independent variable that could affect results. For consistency, we therefore selected only fully-grown oocytes and oocytes that underwent GVBD within 2 h since these are the most meiotically competent. Oocytes were then randomly allocated to either the treatment or control groups. From that point onwards, we needed to keep the two groups separate and identified by label at all stages since there was no other way of being certain that we were reporting on the correct group.