Telomere dysfunction triggers developmentally regulated germ cell apoptosis

Abstract

Telomere dysfunction results in fertility defects in a number of organisms. Although data from fission yeast and Caenorhabditis elegans suggests that telomere dysfunction manifests itself primarily as defects in proper meiotic chromosome segregation, it is unclear how mammalian telomere dysfunction results in germ cell death. To investigate the specific effects of telomere dysfunction on mammalian germ cell development, we examined the meiotic progression and germ cell apoptosis in late generation telomerase null mice. Our results indicate that chromosome asynapsis and missegregation are not the cause of infertility in mice with shortened telomeres. Rather, telomere dysfunction is recognized at the onset of meiosis, and cells with telomeric defects are removed from the germ cell precursor pool. This germ cell telomere surveillance may be an important mechanism to protect against the transmission of dysfunctional telomeres and chromosomal abnormalities.

Figure 1

Representative pachytene, diakinesis, and metaphase I figures from mTR+/+, mTR−/− G5, and mTR−/− G6 air-dried testis spreads. Quantitative data are shown in Table 1. No visible cytogenetic abnormalities were seen in mTR−/− testes (630× magnification).

Figure 2

GCNA1 and TUNEL staining colocalize near the periphery of seminiferous tubules in late generation mTR−/− testis. (A) GCNA1 (red) and TUNEL (green) staining of a seminiferous tubule from a 10-wk-old mTR+/+ mouse testis (630× magnification). (B) GCNA1 (red) and TUNEL (green) staining of a seminiferous tubule from a 10-wk-old mTR−/− G6 mouse testis. White arrows indicate clusters of TUNEL-positive cells.

Figure 3

TUNEL-positive cells localize to the Ku70-negative zone in late generation mTR−/− testes. (A) Ku70 (red) and (B) TUNEL (green) staining of serial sections of a stage II-IV seminiferous tubule. Early meiotic cells at this stage are localized two to three cell layers from the basement membrane of the seminiferous tubule. Ku70-negative and TUNEL-positive cells colocalize to this layer of cells in early meiotic prophase. (C) Ku70 (red) and (D) TUNEL (green) staining of serial sections of a stage VIII-IV seminiferous tubule. Early meiotic cells are localized at the basement membrane of the seminiferous tubule. Ku70-negative and TUNEL-positive cells colocalize to this layer of cells in early meiotic prophase. White arrows indicate TUNEL-positive cells without detectable Ku70 staining. Yellow arrows indicate the few TUNEL-positive cells with detectable Ku70 staining, likely from normal apoptotic processes. Cell nuclei in these tissue sections are counterstained with 4,6-diamidino-2-phenylindole.

Figure 4

Seminiferous tubule atrophy, germ cell apoptosis, and decreased testicular weight are seen at specific developmental stages in late generation mTR−/− mice. (A) Number of TUNEL-positive cells per 100 seminiferous tubule cross sections from mTR+/+ and mTR−/− G6 mice, compared at day 11 and day 13 postpartum. (B) Relative testis weights of mTR+/+ and mTR−/− G6/G7 mice compared at different postpartum developmental ages. (C) Hematoxylin and eosin-stained testis sections from 12–13 d, 6 wk, and 6-mo-old mTR+/+ and mTR−/− G6 mice (400× magnification).

Figure 5

Underrepresentation of germ cells compared with somatic cells that lack telomere signal in postnatal day 22–24 mTR−/− G5 and G6 mice. Metaphases from postnatal day 10 mTR−/− G5 and G6 and postnatal day 22–24 mTR−/− G5 and G6 somatic cells (splenocytes) and germ cells were hybridized with a (TTAGGG)₃ quantitative-FISH probe and scored for the number of chromosome ends without detectable telomere repeats per metaphase. The average from 20 metaphases from each of eight independent postnatal day 10 mTR−/− G5 and G6 or10 independent postnatal day 22–24 mTR−/− G5 and G6 mice is shown.