Lack of response to unaligned chromosomes in mammalian female gametes

Abstract

Chromosome segregation errors are highly frequent in mammalian female meiosis, and their incidence gradually increases with maternal age. The fate of aneuploid eggs is obviously dependent on the stringency of mechanisms for detecting unattached or repairing incorrectly attached kinetochores. In case of their failure, the newly formed embryo will inherit the impaired set of chromosomes, which will have severe consequences for its further development. Whether spindle assembly checkpoint (SAC) in oocytes is capable of arresting cell cycle progression in response to unaligned kinetochores was discussed for a long time. It is known that abolishing SAC increases frequency of chromosome segregation errors and causes precocious entry into anaphase; SAC, therefore, seems to be essential for normal chromosome segregation in meiosis I. However, it was also reported that for anaphase-promoting complex (APC) activation, which is a prerequisite for entering anaphase; alignment of only a critical mass of kinetochores on equatorial plane is sufficient. This indicates that the function of SAC and of cooperating chromosome attachment correction mechanisms in oocytes is different from somatic cells. To analyze this phenomenon, we used live cell confocal microscopy to monitor chromosome movements, spindle formation, APC activation and polar body extrusion (PBE) simultaneously in individual oocytes at various time points during first meiotic division. Our results, using oocytes from aged animals and interspecific crosses, demonstrate that multiple unaligned kinetochores and severe congression defects are tolerated at the metaphase to anaphase transition, although such cells retain sensitivity to nocodazole. This indicates that checkpoint mechanisms, operating in oocytes at this point, are essential for accurate timing of APC activation in meiosis I, but they are insufficient in detection or correction of unaligned chromosomes, preparing thus conditions for propagation of the aneuploidy to the embryo.

Figure 1. The duration of meiosis I is not affected by high frequency of aneuploidy. (A) Aneuploidy frequency in Young CD1 (green, n = 73), Old CD1 (red, n = 47) and Hybrid (gray, n = 33) was scored after monastrol treatment in meiosis II. Image represents typical staining of chromosomes with DAPI (blue), and kinetochores (red). (B) The length of meiosis I - oocytes were microinjected with fluorescently labeled histone H2B (red) and β-tubulin 2A (green) and analyzed by live imaging. Movie frames showing intervals from GVBD until DNA separation, time is indicated for each frame. Lower chart – the average length (hrs) of meiosis I in Young CD1 (green, n = 50), Old CD1 (red, n = 46) and Hybrid (green, n = 21) oocytes, error bars showing SD. (C) Dynamics of securin degradation during meiosis I. Average securin expression levels in Young CD1 (blue, n = 18), Old CD1 (red, n = 21) and Hybrid oocytes (n = 8) are shown, expression levels were normalized to the first frame.

Figure 2. Time-lapse analysis of chromosome alignment and securin degradation. Oocytes were microinjected with fluorescently labeled histone H2B (red), β-tubulin 2A (green) and securin (yellow). Movie frames from representative Young CD1, Old CD1 and Hybrid oocyte are shown, the frame with maximal level of securin is marked with white circle.

Figure 3. Metaphase plate formation vs. APC activation. (A) The highest level of securin is represented by vertical line. Squares are showing time of aligning of all chromosomes on equatorial plane in individual oocytes. Young CD1 (green, n = 19), Old CD1 (red, n = 20) and Hybrid (gray, n = 13). (B) Chart showing frequency of oocytes never forming metaphase plate before anaphase onset - Young CD1 (green, n = 19) 0%, Old CD1 (red, n = 20) 55% and Hybrid (gray, n = 13) 92%.

Figure 4. Correlation between chromosome alignment and securin levels. (A) Typical securin expression curve showing intervals for scoring chromosome alignment: -7 = 120, -6 = 90, -5 = 60, -4 = 30 min to highest securin, -3 = highest securin, -2 = half of the securin level, -1 = one frame before anaphase. (B) Scoring of unaligned chromosomes in meiosis I in intervals described in A – Young CD1 (green, n = 19), Old CD1 (red, n = 20) Hybrid (gray, n = 13). (C) Scoring oocytes with all chromosomes aligned on metaphase II plate during 150 min following anaphase I. Young CD1 (green, n = 30), Old CD1 (red, n = 24) Hybrid (gray, n = 15). (D) % of oocytes with metaphase plate at 18 h - Young CD1 (green, n = 22) 91%, Old CD1 (red, n = 20) 0% and Hybrid (gray, n = 13) 56%.

Figure 5. Severe congression defects in meiosis I in Old CD1 and Hybrid oocytes. (A) Oocytes were microinjected with fluorescently labeled histone H2B (red) and β-tubulin 2A (green). DNA was measured within a rectangle representing 25% of the spindle length. Typical images of oocytes with chromosomes aligned on equatorial plane (left panel) and unaligned (right panel). (B) Scoring of congression defects in meiosis I in Young CD1 (green, n = 10), Old CD1 (red, n = 6) Hybrid (gray, n = 5) oocytes, time of selected intervals relative to anaphase is indicated.