Figure 1.
Dynamics of centromere pairing in mouse spermatocytes.
(A) Dynamics of centromere pairing observed in squashed spermatocytes of wild-type mice at meiotic prophase I. In wild-type spermatocytes the number of CREST foci remained constant as cells progressed from pachytene to diplotene (22.9±0.7 and 22.7±0.9 CREST foci for mid and late diplotene respectively) consistent with centromere pairing. Squashed spermatocytes from Sycp3 knockout mice exhibited increased numbers of CREST foci in very early prophase and Scyp1 knockout mice exhibited elevated CREST foci at pachytene consistent with an absence of centromere pairing. Horizontal lines denote the means and vertical lines denote standard deviations. See Table 1 for summary of means, standard deviation and results of statistical tests. (B) The number of RAP1 signals was used to determine the extent of telomeres association in squashed spermatocytes of wild-type mice at meiotic prophase I. Horizontal lines denote the means and vertical lines denote standard deviations. See Table 1 for summary of means and standard deviation. (C) Quantification of centromere pairing was carried out by scoring the number of homologous chromosomes in spermatocytes spreads at the diplotene stage experiencing central chiasmata, and with paired centromeres but unpaired distal telomeres, paired distal telomeres but unpaired centromeres or absence of both centromere and telomere pairing. Note that centromeres are preferentially paired over telomeres. (D) Centromere pairing was analyzed in surface chromosome spreads of wild-type spermatocytes. The cartoon represents homologous chromosomes at mid-diplotene tethered by chiasmata and paired centromeres. Arrowheads indicate paired centromeres for chromosomes undergoing one exchange. Tailed white arrows indicate the single physical link connecting apparent non-exchange homologous chromosomes. Magnification bar represents 5 µm and correspond to all images but only show in one panel.
Table 1.
Number of centromere and telomere associated foci in spermatocytes.
Table 2.
Co-localization of synaptonemal complex components and centromeres in diplotene.
Figure 2.
Centromeric regions are the last to associate during homologous chromosome synapsis.
(A) Representative surface spread spermatocytes at zygotene and pachytene stages were stained for components of the axial/lateral element (SYCP3), and the transverse filament (SYCP1). CREST staining was used as a marker for centromeres. Arrows indicate unpaired centromeres. X and Y indicate the sex chromosomes. Arrows indicate sites of unpaired centromeres. The scale bar represents 5 µm and corresponds to all images but is only shown in one panel. (B) Magnified zygotene and pachytene chromosomes. Arrows indicate unpaired centromeres (C). Spermatocytes were randomly picked and scored according to extent of pairing and whether regions at the centromere, distal telomere or central chromosome arms were paired. The classification of mid zygotene and late zygotene indicate the average synapsis of all chromosomes in a particular nucleus given the asynchrony of synaptonemal complex assembly. Percentage of synapsis is the total extent of synapsis observed in one individual chromosome. Values are expressed as the total number of chromosomes analyzed at the indicated stage of meiosis. The indicated percentage (%) is the number of chromosomes of an indicated chromosome pairing conformation divided by the total number of chromosomes at an indicated meiotic stage, multiplied by one hundred.
Figure 3.
Synaptonemal complex is retained at paired centromeres.
(A–F) Representative surface spread spermatocytes at diplotene were immunostained with components of the lateral element (SYCP3) and transverse filaments (SYCP1) of the synaptonemal complex. Spermatocytes were also stained with the components of the central element of the synaptonemal complex SYCE1 (G–L), SYCE2 (M–R) and TEX12 (S–X). Arrowheads indicate paired centromeric regions showing persistent staining for components of the synaptonemal complex. CREST staining (purple) was used as a marker for centromeres. Given the asynchrony of synaptonemal complex transverse filament dissolution in spermatocytes at mid-diplotene some chromosomes may retain threads of SYCP1 in the arms. However, for chromosomes at an advanced stage, SYCP1 is preferentially maintained at paired centromeres. The scale bar represents 5 µm and applies to all images except for magnified images of individual chromosomes.
Table 3.
SYCP1 association with paired and unpaired centromeres in diplotene.
Figure 4.
Centromere pairing is absent in Sycp1 knockout spermatocytes.
Surface chromosome spreads of pachytene (A, B) and diplotene (C–H) wild-type and Sycp1 knockout spermatocytes. Note that centromeres (red) in Sycp1 knockout spermatocytes are in close proximity but are not paired. In wild-type spreads most centromeric regions remain paired (arrows, C and H), Sycp1 knockout spermatocytes fail to exhibit paired centromeres (arrows in D). (I–K) Wild-type and Sycp1 knockout spermatocytes were treated with okadaic acid to induce progression to later stages of prophase I. Note that whereas in wild-type spermatocytes (I) centromeric regions remained paired (arrows), in Sycp1 knockout spermatocytes (J, K), centromeres are unpaired (J, arrows). The most advanced spermatocytes show an absence of chromosome association (K). The cartoon represents typical homologous chromosomes at mid diplotene exhibiting paired centromeres in wild-type spermatocytes and non-paired centromeres in Sycp1 knockout mice. Scale bar represents 5 µm and applies to all images except E–H.
Figure 5.
Dynamics of SYCP1 and SYCP3 localization on chromosome arms and centromeres during late prophase I.
Surface chromosome spreads were simultaneously stained for SYCP1 and SYCP3 in order to determine their relative timing of disassembly/dissociation from the synaptonemal complex at later stages of prophase I. The cartoon summarizes the observations. The scale bar represents 5 µm and applies to all panels.
Figure 6.
SYCP1 and SYCP3 are differentially retained on chromosomes as cells progress through meiosis I.
Imaging of whole cells was used to evaluate the distribution of SYCP1, SYCP3 and tubulin. (A–F) After removal from chromosome arms SYCP1 is preferentially retained at paired centromeres in spermatocytes until mid diplotene (panel B, arrows). In a subsequent step (panel c, late diplotene) SYCP1 is removed from paired centromeres before or at nuclear envelope break down. (G–P) SYCP3 is removed from chromosome arms in diplotene but is maintained at paired centromeres until anaphase I. Note the reduction of SYCP3 at chromosome arms (panel i, arrowheads) and accumulation at centromeres after late diplotene (panel i, tailed arrows). After nuclear envelope break down and assembly of the spindle a fraction of SYCP3 bridges the centromeres at anaphase I (panels K, white arrows). Single fluorescent channel images for SYCP1 (panels D–F) and SYCP3 (panels L–P) is also shown. The cartoon summarizes the observations. SYCP1 is shown in black. The scale bar represents 5 µm and applies to all panels.
Figure 7.
The coordination of centromere pairing and chromosome segregation at meiosis I.
(A) Model for the role of SYCP1 and SYCP3 in centromere pairing. SYCP1 and SYCP3 play different roles in meiotic chromosome segregation while cohering homologous centromeres. (B) Based on the similarities between mice and other model organism such budding yeast and Drosophila, in which a correlation between late centromere pairing and improved segregation of homologous chromosomes has been demonstrated, we propose that in the absence of genetic exchange, centromere pairing holds homologous centromeres together and facilitates orientation of the homologous chromosomes for movement to the opposite poles during meiosis I division.