The Meiotic Nuclear Lamina Regulates Chromosome Dynamics and Promotes Efficient Homologous Recombination in the Mouse

42Citations
Citations of this article
93Readers
Mendeley users who have this article in their library.

Abstract

The nuclear lamina is the structural scaffold of the nuclear envelope and is well known for its central role in nuclear organization and maintaining nuclear stability and shape. In the past, a number of severe human disorders have been identified to be associated with mutations in lamins. Extensive research on this topic has provided novel important clues about nuclear lamina function. These studies have contributed to the knowledge that the lamina constitutes a complex multifunctional platform combining both structural and regulatory functions. Here, we report that, in addition to the previously demonstrated significance for somatic cell differentiation and maintenance, the nuclear lamina is also an essential determinant for germ cell development. Both male and female mice lacking the short meiosis-specific A-type lamin C2 have a severely defective meiosis, which at least in the male results in infertility. Detailed analysis revealed that lamin C2 is required for telomere-driven dynamic repositioning of meiotic chromosomes. Loss of lamin C2 affects precise synapsis of the homologs and interferes with meiotic double-strand break repair. Taken together, our data explain how the nuclear lamina contributes to meiotic chromosome behaviour and accurate genome haploidization on a mechanistic level. © 2013 Link et al.

Figures

  • Figure 1. Generation and characterization of a lamin C2-deficient mouse line. (A) Murine somatic lamins A and C are composed of exons 1 through 12 and 1 through 10 of the Lmna gene, respectively, both excluding exon 1a. Lamin C2 is encoded by exons 1a through 10 with exon 1a serving as an alternative starting exon specific for lamin C2 [18]. Exon 1a was targeted for construction of a knockout mouse model deficient for lamin C2. SmaI restriction sites and the probe used for southern blot analyses are indicated. (B) Southern blot analyses of genomic mouse DNA digested by SmaI distinguishing wildtype, heterozygous and homozygous lamin C2-deficient animals. (C, C9) RT-PCR and immunoblot analyses showing presence of lamin C2 in the testes of wildtype and heterozygous mice, but absence in lamin C22/2 testes: expression of lamin A/C remained unaffected in all three genetic backgrounds. (C0, C90) Immunohistochemical analyses using anti-A-type lamin antibodies on liver and testis tissues showing unaffected expression of A-type lamins in somatic cells, while in meiotic cells of lamin C22/2 testes A-type lamins are completely absent. (D) Histological sections of wildtype and lamin C22/2 testes demonstrating the absence of post-meiotic cells in lamin C22/2 tissue. Scale bars 100 mm. As shown in the insets, testes size of lamin C22/2 males is significantly reduced. Scale bars 1 mm. S somatic cells, Sg spermatogonia, Sc spermatocytes, Sp spermatids. doi:10.1371/journal.pgen.1003261.g001
  • Figure 2. Defective synapsis of the homologous chromosomes in lamin C22/2 meiocytes. Representative chromosome spreads of spermatocytes (A) and oocytes (17.5 dpf) (B) labelled with anti-SYCP3 and anti-SYCP1 antibodies. Heterologous associations (red box in A9) are observed in lamin C22/2 spermatocytes only. Incomplete pairing of homologs (white boxes in A9 and B9) as well as univalent chromosomes (arrowheads in A0,A90 and B0) occur in both lamin C22/2 spermatocytes and oocytes. Lamin C22/2 spermatocytes also display cells with X and Y chromosomes as the only univalents (arrowheads in A90) or with linear telomere-telomere associations between non-homologous chromosomes (arrow in A00). Scale bars 10 mm. (C) Quantification of meiocytes with defective synapsis in lamin C22/2 males and females. For both sexes, differences between mutants and controls are highly significant (Pearson’s Chi2 test p-value,0.0001 each) (D) Synaptic pairing defects in males were further categorised and quantified. Interestingly, sex chromosomes were univalent in the vast majority of mutant spermatocytes. See text for further discussion. doi:10.1371/journal.pgen.1003261.g002
  • Figure 3. Loss of lamin C2 has no effect on meiotic telomere attachment. (A) 3D-preserved swab preparations showing wildtype (A) and knockout (A9) spermatocytes simultaneously labelled with anti-TRF1 and SUN1 antibodies. As in the wildtype, in lamin C22/2 spermatocytes virtual all telomeres appear to be attached to the NE as indicated by co-localisation of TRF1 and SUN1 signals. Scale bars 5 mm. (B) Quantifications of colocalised and non-co-localised TRF1/SUN1 signals (see A) revealed that ratios of co-localised to non-co-localised spots comparing wildtype and knockout spermatocytes show no significant difference (wildtype n = 33; lamin C22/2 n = 45; Pearson’s Chi2 test p-value: 0.799). (C,D) Chromosome spread preparations of pachytene-like lamin C22/2 spermatocytes showing that all telomeres are associated with SUN1. In (C) TeloFISH and in (D) anti-SUN1 staining in co-localisation with SYCP3. Scale bars 10 mm. doi:10.1371/journal.pgen.1003261.g003
  • Figure 4. Bouquet release is significantly delayed in lamin C22/2 spermatocytes. (A) Representative wildtype and knockout spermatocytes from testes of 10 to 14 day old mice labelled for SYCP3 and SUN1 with clusters of telomeres indicated by asterisks. Scale bars 5 mm. (B) Z-projections of entire nuclei from wildtype and knockout littermates, labelled again for SYCP3 and SUN1, demonstrating the distinguishable difference between clustered and non-clustered telomere patterns; asterisks indicate telomere clusters. Scale bar 5 mm. (C) Quantitative evaluation of spermatocytes showing clustered telomeres in wildtype and knockout littermates aged 10 to 14 dpp. Note the highly significant differences at ages 13 and 14 dpp. Values represent means (6 s.d.) of data from three littermate pairs of wildtype and knockout animals each, separately analysed using Pearson’s Chi2 tests: d10 p = 0.28 (wt, n = 517; lamin C22/2, n = 580); d11 p = 0.15 (wt, n = 677; lamin C22/2, n = 703); d12 p = 0.29 (wt, n = 736; lamin C22/2, n = 780); d13 p = 6.5 x1023 (wt, n = 639; lamin C22/2, n = 648); d14 p = 7.6 x1027 (wt, n = 700; lamin C22/2, n = 682). ***p,0.01 (D) Representative wildtype and lamin C22/2 spermatocytes from day 14 aged mice showing comparable stages of synaptonemal complex assembly as indicated by SYCP1 labelling. Arrowheads denote homologous pairing defects in the absence of lamin C2; the asterisks indicate telomere clustering. Scale bar 5 mm. doi:10.1371/journal.pgen.1003261.g004
  • Figure 5. Alterations of meiotic DNA double-strand break (DSB) repair and inefficient homologous recombination caused by lamin C2 deficiency. Chromosome spread preparations were double-stained using anti-SYCP3 antibodies in combination with anti-cH2AX (A,E), antiRAD51 (B), anti-RPA (C) or anti-MLH1 (D,F) antibodies. (A–D) In males, lamin C22/2 spermatocytes show incomplete repair of induced DSB as demonstrated by the increased persistence of cH2AX (A9), RAD51 (B9) and RPA (C9) and they lack crossing over events indicated by the complete absence of MLH1 (D9). (E) In females, efficient DSB repair is also affected in lamin C2-deficient pachytene oocytes (17.5 dpf) as revealed by intensive cH2AX staining surrounding incompletely synapsed chromatin (asterisk in E9). (F) In contrast to males, lamin C22/2 females are able to recruit MLH1 onto chromosome axes in 19.5 dpf late-pachytene oocytes. However, the MLH1 foci number was lowered and a significant portion of cells lacked at least one obligate MLH1 focus indicating the absence of cross over recombination on the affected chromosomes (arrowheads in F9 and F0). This phenomenon was likewise seen in oocytes with (asterisk in F0) or without (F9) obvious synaptic defects. Scale bars 10 mm. (G) Quantification of pachytene oocytes with persistence of cH2AX in wildtype and knockout littermates reveals inefficient DSB repair in lamin C2-deficient cells (Pearson’s Chi2 test p-value = 0.0323). (H) Whisker box plot showing the number of MLH1 foci per cell in late pachytene oocytes of lamin C22/2 and control (lamin C2+/+ and lamin C2+2/) animals. Two pairs of littermates were analysed. Knockout oocytes show a significant reduction of MLH1 foci number, indicating a lowered rate of homologous recombination (median foci numbers: control, 29; lamin C22/2, 25.5; Mann-Whitney U-test pvalue = 0.00025). (I) The number of late pachytene oocytes lacking one or more obligate MLH1 foci is significantly elevated in lamin C22/2 animals compared to the controls (lamin C2+/+ and lamin C2+/2). As above, two pairs of littermates were analysed (Pearson’s Chi2 test p-value = 0.0005). doi:10.1371/journal.pgen.1003261.g005

References Powered by Scopus

Coupling of the nucleus and cytoplasm: Role of the LINC complex

1056Citations
N/AReaders
Get full text

Loss of A-type lamin expression compromises nuclear envelope integrity leading to muscular dystrophy

1030Citations
N/AReaders
Get full text

Recombinational DNA double-strand breaks in mice precede synapsis

738Citations
N/AReaders
Get full text

Cited by Powered by Scopus

Mechanisms and functions of nuclear envelope remodelling

258Citations
N/AReaders
Get full text

Accessorizing and anchoring the LINC complex for multifunctionality

225Citations
N/AReaders
Get full text

Polar body genome transfer for preventing the transmission of inherited mitochondrial diseases

142Citations
N/AReaders
Get full text

Register to see more suggestions

Mendeley helps you to discover research relevant for your work.

Already have an account?

Cite

CITATION STYLE

APA

Link, J., Jahn, D., Schmitt, J., Göb, E., Baar, J., Ortega, S., … Alsheimer, M. (2013). The Meiotic Nuclear Lamina Regulates Chromosome Dynamics and Promotes Efficient Homologous Recombination in the Mouse. PLoS Genetics, 9(1). https://doi.org/10.1371/journal.pgen.1003261

Readers over time

‘13‘14‘15‘16‘17‘18‘19‘20‘21‘22‘23‘240481216

Readers' Seniority

Tooltip

PhD / Post grad / Masters / Doc 41

60%

Researcher 21

31%

Professor / Associate Prof. 5

7%

Lecturer / Post doc 1

1%

Readers' Discipline

Tooltip

Agricultural and Biological Sciences 41

55%

Biochemistry, Genetics and Molecular Bi... 25

33%

Medicine and Dentistry 8

11%

Chemistry 1

1%

Save time finding and organizing research with Mendeley

Sign up for free
0