Cyclin B2 is required for progression through meiosis in mouse oocytes

Abstract

Cyclins associate with cyclin-dependent serine/threonine kinase 1 (CDK1) to generate the M phase-promoting factor (MPF) activity essential for progression through mitosis and meiosis. Although cyclin B1 (CCNB1) is required for embryo development, previous studies concluded that CCNB2 is dispensable for cell cycle progression. Given previous findings of high Ccnb2 mRNA translation rates in prophase-arrested oocytes, we re-evaluated the role of this cyclin during meiosis. Ccnb2^-/- oocytes underwent delayed germinal vesicle breakdown and showed defects during the metaphase-to-anaphase transition. This defective maturation was associated with compromised Ccnb1 and Moloney sarcoma oncogene (Mos) mRNA translation, delayed spindle assembly and increased errors in chromosome segregation. Given these defects, a significant percentage of oocytes failed to complete meiosis I because the spindle assembly checkpoint remained active and anaphase-promoting complex/cyclosome function was inhibited. In vivo, CCNB2 depletion caused ovulation of immature oocytes, premature ovarian failure, and compromised female fecundity. These findings demonstrate that CCNB2 is required to assemble sufficient pre-MPF for timely meiosis re-entry and progression. Although endogenous cyclins cannot compensate, overexpression of CCNB1/2 rescues the meiotic phenotypes, indicating similar molecular properties but divergent modes of regulation of these cyclins.

Keywords: Cyclins; Fertility; MPF; Meiosis; Mouse oocytes.

Fig. 1.

Translation of Ccnb1 and Ccnb2 mRNAs is differentially regulated during meiotic maturation in mouse oocytes. (A,B) RNA-Seq was performed using mRNA extracts from total cell lysate (total mRNA) or after immunoprecipitation of HA-tagged ribosomes (ribosome-bound mRNA) from oocytes arrested in prophase with cilostamide (time 0) or collected 2, 4, 6 and 8 h after meiotic resumption. Counts per million (CPM) of mapped reads are reported for Ccnb1 (A) and Ccnb2 (B); average CPMs of two independent biological replicates with range are reported. (C) Poly(A) tail lengths of the Ccnb1 and Ccnb2 mRNAs in GV oocytes. The data were mined from PMID: 28792939 and are reported as binned values of up to 80 (A) nucleotides. (D) Rates of translation of Ccnb1 and Ccnb2 mRNA variants in prophase I. Oocytes were injected with a 1:1 mix of oligo-adenylated YFP-(Ccnb2-short, Ccnb2-long, Ccnb1-short or Ccnb1-long) 3′UTR and polyadenylated mCherry. Rate of translation in GV-arrested oocytes were calculated with a 3 h window at a sampling rate of 15 min. Student's t-tests were performed for statistical significance; ***P<0.0001. (E) Translational efficiency of Ccnb1 and Ccnb2 mRNA was calculated by dividing the CPMs of ribosome-bound mRNA by the CPMs of total mRNA. Four biological replicates were used for these calculations. (F) To evaluate the absolute concentration of cyclins in GV-arrested oocytes, western blots were performed using cell lysates and cyclin levels were quantified by interpolating from a standard curve of known concentrations of CCNB1 and CCNB2 recombinant proteins. Calculated concentrations are reported as the mean and s.d. of three independent biological replicates. ns, not significant.

Fig. 2.

Ccnb2^−/− mice show compromised fecundity. (A) Cumulative number of pups per female derived from different mating schemes. Mating schemes and number of pairs were as follows: +/+♂×+/+♀, n=20; +/−♂×+/−♀, n=35; −/−♂×−/−♀, n=6; +/+♂×−/−♀, n=6. Student's t-tests were performed between +/−♂×+/−♀ and −/−♂×−/−♀ (red asterisks) or +/−♂×+/−♀ and +/+♂×−/−♀ (blue asterisks); each point is the mean±s.e.m. of the number of pups obtained; **P<0.01, ***P<0.001. Breeding was initiated when the mice reached 8 weeks of age. (B) Pup body weights from +/−♂×+/−♀ matings were recorded 21 days after birth. The weight of each mouse was normalized for the average weight of the litter and plotted according to their genotype as medians with interquartile range. Student's t-tests were performed for statistical significance; ***P<0.0002. (C) Representative histological sections of ovaries from Ccnb2^+/+ and Ccnb2^−/− mice. Follicle stages were scored and representative pictures of pre-antral, early antral (when multiple cavities have not coalesced in a single antrum), late antral (when a single, large antrum is present) follicles and corpora lutea are shown. (D) Follicle counts were performed on pairs of ovaries from three biological replicates for each group and are plotted as mean±s.d. ns, not significant.

Fig. 3.

Aberrant timing of meiotic resumption in oocytes depleted of CCNB2 results from defective pre-MPF. (A) Western blot performed on lysates of 150 oocytes from Ccnb2^+/+, ^+/− and ^−/− mice. (B) Kinase assays were performed using increasing numbers of oocytes from Ccnb2^+/+ (+/+) or Ccnb2^−/− (−/−) mice and a GST-PP1 fragment as a substrate. T320 PP1 phosphorylation was detected by a phospho-specific antibody (pT320-PP1). The level of total substrate loaded was evaluated by Ponceau S staining (Total-PP1). (C) Quantification of six independent kinase assays. pT320-PP1/Total-PP1 ratios from Ccnb2^−/− oocytes were expressed as fold changes over their matched Ccnb2^+/+ controls. Signals are expressed as median with interquartile range. A Student's t-test was performed to determine statistical significance; ***P=0.0007. (D) Time of GVBD was determined through brightfield images acquired every 15 min up to 24 h. Times are plotted as medians with interquartile ranges, averages (Av), s.d. and number of oocytes (n) observed from seven independent experiments. A nonparametric Mann–Whitney test was performed to evaluate statistical significance; ****P<0.0001. (E) Oocytes were injected with mRNA encoding either Ccnb1-mCherry or Ccnb2-mCherry and, after 3 h incubation, were released in cilostamide-free medium. GVBD time and statistical significance were determined as in (D); times are reported as medians with interquartile ranges, Av, s.d. and number of oocytes (n) scored from two independent experiments. ns, not significant.

Fig. 4.

The rate of WEE1B but not CDC25 translocation is decreased on CccnB2^−/− oocytes. Oocytes were injected with inactive Cdc25B-YFP (A) or Wee1B-YFP (B) and, after overnight incubation, were released in cilostamide-free medium. Brightfield and YFP images were acquired every 5 min for 20 h. (A,B) Images of a Ccnb2^+/+ oocyte and of two examples of Ccnb2^−/− oocytes. The red box marks the time of GVBD. Scale bars: 20 µm. (C) Rates of CDC25-YFP or (D) WEE1B-YFP translocation were calculated from individual oocytes as the slope of the linear regression of the nuclear/cytoplasmic ratios. Rates are expressed as medians with interquartile range and the number of oocytes from two independent experiments is reported for each condition. Student's t-tests were performed to assess statistical significance; ****P<0.0001. ns, not significant.

Fig. 5.

MI spindle formation and activation of Ccnb1 and Mos translation are disrupted in Ccnb2^−/− mice. (A,B) Oocytes were injected with a 1:1 mix of polyadenylated mCherry and either YFP-Ccnb1-long 3′UTR (A) or YFP-Mos 3′UTR (B). After overnight incubation, oocytes were released in cilostamide-free medium, and brightfield, YFP and mCherry images were acquired every 15 min for 24 h. YFP signals were normalized by maximal mCherry signals (YFP/mCherry). The normalized rate of YFP accumulation was calculated for each oocyte before (0-2 h) and after (4-6 h) GVBD. Rates were plotted as the median (red) and interquartile range. Student's t-tests were used to evaluate statistical significance; **P=0.0058, ****P<0.0001. The number of oocytes from three independent experiments is reported for each condition. (C) Oocytes were released in cilostamide-free medium and fixed for 8 h after meiotic resumption. The spindle, chromatin and kinetochores were visualized with β-tubulin 488 antibody, DAPI and CREST, respectively. Images show oocytes arrested in prophase I, GVBD without a spindle, early spindle (tubulin organized into a sphere surrounded by chromosomes) and bipolar MI spindle. Scale bars: 10 µm. (D) Oocytes were scored for maturation stage and data are presented as the percentage of total oocytes. The number of oocytes from two independent experiments observed for each group is reported.

Fig. 6.

A subpopulation of Ccnb2^−/− oocytes fails to complete meiosis I because of altered APC activation. Oocytes were released in cilostamide-free medium and brightfield images were captured every 15 min. (A) The cumulative PBE times were plotted and Student's t-tests between Ccnb2^+/+ and Ccnb2^−/− oocytes performed (red asterisks); *P<0.05, **P<0.01. Bars represent the s.e.m.; the number of oocytes from seven independent experiments are reported. (B,C) Oocytes were released in cilostamide-free medium and fixed after 24 h. The spindle and the chromatin were visualized with β-tubulin 488 antibody and DAPI, respectively. (B) Images of oocytes arrested in prophase I, MI, telophase I and MII. Scale bars: 10 µm. (C) Oocytes were scored for maturation stage [reported in (B)] and data were reported as the percentage of total oocytes from three independent experiments. (D) Oocytes were injected with mRNA encoding the APC substrate Securin-YFP and, after 17 h incubation, released in cilostamide-free medium. The securin-YFP level was measured every 15 min. Oocytes from three independent experiments were observed. (E,F) Oocytes were injected with mRNA encoding either Ccnb1-mCherry (E) or Ccnb2-mCherry (F) and, after 3 h incubation, released in cilostamide-free medium. CCNB1-mCherry or CCNB2-mCherry levels were measured every 15 min. Oocytes from three and two independent experiments were observed, respectively.

Fig. 7.

A population of Ccnb2^−/− oocytes arrests in MI because of persistent SAC activity. (A,B) Oocytes were released in cilostamide-free medium and fixed at the indicated times. Where specified, oocytes were treated with nocodazole 15 min before fixation. MAD2 and CREST, and chromatin were visualized with specific antibodies and DAPI, respectively. (A) Representative pictures are presented for each condition. Scale bars: 10 µm (B) The amount of MAD2 localized at each kinetochore was quantified by measuring the MAD2/CREST ratio. Signals are plotted as the mean±s.e.m. The number of kinetochores analyzed from two independent experiment is reported. Student's t-tests were used to evaluate statistical significance; ns: not significant, **** indicates P<0.0001. (C) Oocytes were released in the absence or presence of 100 nM reversine. The time of GVBD and PBE were determined through brightfield images acquired every 15 min for 24 h. The length of meiosis I was calculated as the time between GVBD and PBE. Five independent experiments are included. (D,E) Oocytes were matured for 20 h with or without 100 nM reversine. In situ chromosome spreads were performed by treating oocytes for 2 h with 100 µM monastrol. Oocytes were then fixed and immunofluorescence stained for kinetochores using CREST (green) and for chromosomes using DAPI (red). Confocal pictures were obtained every 0.2 µm and maximal intensity projections were produced with ImageJ. Kinetochores were quantified throughout the Z-stacks Scale bars: 5 µm. (E) Two independent experiments were analyzed and Fisher exact tests were used to evaluate statistical significance; *P<0.05.