A spindle assembly checkpoint protein functions in prophase I arrest and prometaphase progression

Abstract

Two critical stages of mammalian oocyte regulation are prophase I arrest, which is important for sustaining the oocyte pool, and the progression through meiosis I (MI) to produce fertilizable eggs. We have found that the spindle assembly checkpoint protein BubR1 regulates both stages in mouse oocytes. We show that oocytes depleted of BubR1 cannot sustain prophase I arrest and readily undergo germinal vesicle breakdown, a marker for reentry into MI. BubR1-depleted oocytes then arrest before completing MI, marked by failure of polar body extrusion. Both meiotic defects in BubR1-depleted oocytes are due to reduced activity of the master regulator known as the anaphase-promoting complex (APC), brought about through diminished levels of the APC coactivator Cdh1.

Fig. 1

Prophase I arrest is compromised after BubR1-depletion. (A) Schematic of MI. (B) Mock-depleted (+ControlMO), BubR1-depleted (+BubR1MO), Mad2-depleted (+Mad2MO)(7), Cdh1-depleted (+Cdh1MO)(9) and uninjected oocytes were scored for GVBD after 24 h in IBMX. Note that Cdh1-depleted oocytes escape prophase I arrest as shown previously(2). Increased GVBD following BubR1-depletion could be prevented by expressing either BubR1 from an hBubR1 cRNA (+BubR1MO+hBubR1 cRNA) or Cdh1 from a Cdh1 cRNA (+BubR1MO+Cdh1 cRNA)(9). Error bars, mean ± SEM; N ≥ 3. Asterisks denote a significant difference from uninjected controls (P < 0.0001; Student’s t-test). (C and D) Samples (50 oocytes) of GV-stage oocytes from each of the groups depicted were immunoblotted either for Cdh1 and actin (C) or for BubR1, Cdh1 and GAPDH (D) (N = 2). (*), non-specific band.

Fig. 2

BubR1-depletion induces a prometaphase I arrest. (A) Timeline of PBE for uninjected controls, Mad2-depleted and BubR1-depleted oocytes. Oocytes were scored for the presence of polar bodies at 6 h, 7 h , 8 h, 9 h and 10 h post-GVBD. Note the contrasting effects of Mad2- and BubR1-depletion on PBE. (B) PBE is inhibited after BubR1-depletion. PBE rates were determined at 10 h and 20 h post-GVBD. Note that prometaphase I arrest is robust after BubR1-depleti on so that PBE increases only modestly (from 6% to 11%) during an additional 10 h of culture (from 10 h post-GVBD to 20 h post-GVBD) . Furthermore, co-depletion of Mad2 does not restore PBE rates indicating that MI arrest is not SAC-mediated. Error bars, me an + SEM; N ≥ 3. Asterisks denote a significant difference from uninjected controls (P < 0.0001; Student’s t test). (C) Samples (50 oocytes) of BubR1-depleted oocytes at 2 h, 4 h, 6 h and 8 h post-GVBD were blotted for cyclin B1, securin and actin. (D and E) Control and BubR1-depleted oocytes were immunostained for tubulin and DNA at the times post-GVBD indicated in the figure (n ≥ 15 oocytes per time-point). Note that by 10 h post-GVBD in controls, a polar body (white arrowhead) with associated chromosomes is present.

Fig. 3

(A to C) Cdh1 is reduced after BubR1-depletion. Samples (30 oocytes) of BubR1-depleted oocytes were immunoblotted along with uninjected controls at 3 h, 6 h and 9 h post-GVBD for Cdh1, Cdc20 and actin (A). Band intensities of (B) Cdh1 and (C) Cdc20 on blots were quantified and normalised to values found in controls. (D to F) Securin is preferentially stabilised after either BubR1- or Cdh1-depletion. Samples (30 oocytes) of BubR1-depleted and Cdh1-depleted oocytes were immunoblotted along with uninjected controls at 3 h and 6 h post-GVBD for securin, cyclin B1 and GAPDH (D). Band intensities of (E) securin and (F) cyclin B1 on blots were quantified and normalised to values found in controls. Error bars, mean + SEM; N = 3. Asterisks (B and C and E) denote a significant difference from uninjected controls (P < 0.0001; Student’s t test).

Fig. 4

BubR1-depletion impairs kinetochore-microtubule attachments. (A to C) At 8 h post-GVB D, control oocytes (A) and BubR1-depleted oocytes (B and C) were immunostained for tubulin, DNA and Mad2. (D to F) Control oocytes were cold-treated(9) and immunostained at 4 h post-GVBD (D) and 8 h post-GVBD (E). Note the change in spindle morphology afte r cold-treatment (compare A and E). BubR1-depleted oocytes were cold-treated and immunostained at 8 h post-GVBD (F) (n ≥ 20 oocytes per group). Note that chromosomes are extended although kinetochore-microtubule attachments are lacking (B and C), implicating direct contacts between microtubules and chromatin as demonstrated previously(15). Conversely, chromosomes become compacted when microtubules are lacking (F, white arrow).