Atm inactivation results in aberrant telomere clustering during meiotic prophase

Abstract

A-T (ataxia telangiectasia) individuals frequently display gonadal atrophy, and Atm-/- mice show spermatogenic failure due to arrest at prophase of meiosis I. Chromosomal movements take place during meiotic prophase, with telomeres congregating on the nuclear envelope to transiently form a cluster during the leptotene/zygotene transition (bouquet arrangement). Since the ATM protein has been implicated in telomere metabolism of somatic cells, we have set out to investigate the effects of Atm inactivation on meiotic telomere behavior. Fluorescent in situ hybridization and synaptonemal complex (SC) immunostaining of structurally preserved spermatocytes I revealed that telomere clustering occurs aberrantly in Atm-/- mice. Numerous spermatocytes of Atm-/- mice displayed locally accumulated telomeres with stretches of SC near the clustered chromosome ends. This contrasted with spermatogenesis of normal mice, where only a few leptotene/zygotene spermatocytes I with clustered telomeres were detected. Pachytene nuclei, which were much more abundant in normal mice, displayed telomeres scattered over the nuclear periphery. It appears that the timing and occurrence of chromosome polarization is altered in Atm-/- mice. When we examined telomere-nuclear matrix interactions in spermatocytes I, a significant difference was observed in the ratio of soluble versus matrix-associated telomeric DNA sequences between meiocytes of Atm-/- and control mice. We propose that the severe disruption of spermatogenesis during early prophase I in the absence of functional Atm may be partly due to altered interactions of telomeres with the nuclear matrix and distorted meiotic telomere clustering.

FIG. 1

Giemsa-stained spermatocytes of 42-day-old mice. (a) Control mice show meiosis metaphase I along with other well-differentiated cells of spermatocytes. Arrow denotes bivalents at metaphase I. (b) Preparation from an Atm^−/− testicle. Note the absence of condensed bivalents or mature spermatocytes.

FIG. 2

Spreads of spermatocyte after SC immunostaining with anti-SCP3 antiserum. (a) SC of pachytene spermatocytes of control mice. The arrow indicates the sex vesicles in control spermatocytes. (b) Atm^−/− spermatocytes show aberrant synapsis with unpaired axial cores, nonhomologous synapsis, and fragmented SCs.

FIG. 3

SCP3 immunostaining (yellowish) to structurally preserved spermatocytes from normal (a to c) and Atm^−/− (d to f) testicle suspensions. DAPI images (gray) are shown to the right. (a) Zygotene nucleus with partial synapsis. (b and c) Pachytene nuclei with variously arranged SCs. (d) Atm^−/− spermatocyte nucleus with a prominent chromocenter (seen as white mass in the DAPI image) and fragments of SC located at the chromocenter (compare with Fig. 5c and d). (e) Atm^−/− spermatocyte with SCs looping out from the single chromocenter (bouquet arrangement). (f) Aberrant Atm^−/− spermatocyte with long axial elements and few stretches of SCs.

FIG. 4

FISH with chromosome 8-specific proximal probes to Atm^−/− spermatocytes. (a) Nonaligned, with two signals; (b) aligned with two signals in close proximity; (c) fused signals.

FIG. 5

Spermatocytes showing telomere clustering and subsequent separation of telomeres in postleptotene stage of meiosis prophase I as detected by the TTAGGG probe and propidium iodide used as counterstain. (a and b) Spermatocytes of control mice at leptotene/zygotene stage. (c) Spermatocyte of Atm^−/− mice at leptotene/zygotene stage. Note that panels a to c have the fewest telomere signals but each signal is created by numerous closely spaced signals. Panels d to f show increased telomere signal numbers in cells from control mice with disperse telomere arrangement, as the clustered telomeres have organized in proper pairs. Such telomere dispersal at pachytene was rarely seen in Atm^−/− spermatocytes.

FIG. 6

Telomere FISH (green) and simultaneous SCP3 immunostaining (red) to structurally preserved bouquet spermatocytes of normal (a and b) and Atm^−/− (c and d) spermatocyte nuclei. DAPI (blue) marks the nuclear outline. (a) Leptotene/early zygotene nucleus with partially clustered telomeres and numerous axial core fragments; (b) zygotene nucleus with partially clustered telomeres and extensive synapsis; (c) polarized Atm^−/− spermatocyte with clustered telomeres and aberrant large SC-protein signals at and near the telomeres (yellow indicates color overlap); (d) Atm^−/− spermatocyte nucleus with clustered telomeres and aberrant synapsis (arrow denotes an SC stretch which connects at least three telomere-proximal axial cores).

FIG. 7

Autoradiograph visualizing telomeric DNA. Halo preparations were digested with StyI and centrifuged to separate S and P telomeric sequences. Lanes show total (T; P plus S), S, and P telomeric DNA fractions in spermatogonia of Atm null (a) and control (b) mice. Lanes P and S for control mice and lanes P and S for Atm null mice represent telomeric DNA from similar numbers of halos. Note the difference in the P/S fraction ratio between Atm null and control mouse cells.