Meiotic Telomere Distribution and Sertoli Cell Nuclear Architecture Are Altered in Atm- andAtm-p53-Deficient Mice

Abstract

The ataxia telangiectasia mutant (ATM) protein is an intrinsic part of the cell cycle machinery that surveys genomic integrity and responses to genotoxic insult. Individuals with ataxia telangiectasia as well as Atm ؊/؊ mice are predisposed to cancer and are infertile due to spermatogenesis disruption during first meiotic prophase. Atm ؊/؊ spermatocytes frequently display aberrant synapsis and clustered telomeres (bouquet topology). Here, we used telomere fluorescent in situ hybridization and immunofluorescence (IF) staining of SCP3 and testes-specific histone H1 (H1t) to spermatocytes of Atm-and Atm-p53-deficient mice and investigated whether gonadal atrophy in Atm-null mice is associated with stalling of telomere motility in meiotic prophase. SCP3-H1t IF revealed that most Atm ؊/؊ p53 ؊/؊ spermatocytes degenerated during late zygotene, while a few progressed to pachytene and diplotene and some even beyond metaphase II, as indicated by the presence of a few round spermatids. In Atm ؊/؊ p53 ؊/؊ meiosis, the frequency of spermatocytes I with bouquet topology was elevated 72-fold. Bouquet spermatocytes with clustered telomeres were generally void of H1t signals, while mid-late pachytene and diplotene Atm ؊/؊ p53 ؊/؊ spermatocytes displayed expression of H1t and showed telomeres dispersed over the nuclear periphery. Thus, it appears that meiotic telomere movements occur independently of ATM signaling. Atm inactivation more likely leads to accumulation of spermatocytes I with bouquet topology by slowing progression through initial stages of first meiotic prophase and an ensuing arrest and demise of spermatocytes I. Sertoli cells (SECs), which contribute to faithful spermatogenesis, in the Atm mutants were found to frequently display numerous heterochromatin and telomere clusters-a nuclear topology which resembles that of immature SECs. However, Atm ؊/؊ SECs exhibited a mature vimentin and cytokeratin 8 intermediate filament expression signature. Upon IF with ATM antibodies, we observed ATM signals throughout the nuclei of human and mouse SECs, spermatocytes I, and haploid round spermatids. ATM but not H1t was absent from elongating spermatid nuclei. Thus, ATM appears to be removed from spermatid nuclei prior to the occurrence of DNA nicks which emanate as a consequence of nucleoprotamine formation.Ataxia telangiectasia is a rare human recessive autosomal disorder with a pleiotropic phenotype, specifically including progressive neurological degeneration, telangiectasia, often associated with premature aging, reduced size, immunodeficiency, sensitivity to ionizing radiation, cancer predisposition, and infertility (13,40,81,93). Mice mutated in the homologue of the ATM (ataxia telangiectasia mutated) gene (Atm) display similar pleiotropic defects (5, 27, 97). The ATM protein belongs to a growing family of phosphatidylinositol-3 kinaserelated kinases and seems to play a role as an intrinsic part of the cell cycle machinery that surveys genomic integrity, cell cycle progression, and processing of DNA damage. It shows sim...

ATM function and telomere stability

“…One possible interpretation for the accumulation of spermatocytes I with bouquet topology in Atm de®cient cells could be slow progression through initial stages of ®rst meiotic prophase and an ensuing arrest and demise of spermatocytes I. Sertoli cells contribute to faithful spermatogenesis. In the Atm-mutants, sertoli cells were found to frequently display numerous heterochromatin-and telomere clusters ± a nuclear topology which resembles that of immature sertoli cells (Scherthan et al, 2000). Atm disruption causes an immature nuclear architecture/heterochromatin distribution in sertoli cells, the supportive somatic cell lineage of the seminiferous epithelium; which display strong immunouorescent Atm signals in their chromatin.…”

“…Recent studies have shown that ATM is associated with chromatin (Gately et al, 1998;Scherthan et al, 2000) and a fraction of the ATM is detected in nuclear aggregates (Andegeko et al, 2001). The striking correlation between the appearance of retained ATM and of histone H2AX (?-H2AX), and the rapid association of a fraction of ATM with ?-H2AX foci, are consistent with a major role for ATM in the early detection of DSBs and subsequent induction of cellular responses.…”

MOF and Histone H4 Acetylation at Lysine 16 Are Critical for DNA Damage Response and Double-Strand Break Repair

“…After air drying, slides were stored at Ϫ20°C until further use. Immunofluorescent staining was done as described previously (24,67).…”

hTERT associates with human telomeres and enhances genomic stability and DNA repair

“…Telo-FISH was combined with IF. This was achieved by first performing immunostaining and then preparations were subjected to simultaneous denaturation in the presence of the telomere probe (Scherthan et al, 2000). Labeling, FISH and detection of oligonucleotides were performed as described (Scherthan et al, 2000).…”

“…Generally, mice of about 4 months of age, were killed and testes resected for further processing or instant snap freezing in liquid nitrogen. Structurally preserved suspension nuclei were prepared by crosslinking fixation with PBS-buffered formaldehyde (Scherthan et al, 2000). A polyclonal rabbit anti-SCP3 antiserum was utilized to detect axial cores and complete SCs .…”