Distribution of Atr protein in primary spermatocytes of a mouse chromosomal mutant: a comparison of preparation techniques

In this study, we examined the suitability of a three dimensional preparati on technique for studying chromosome behaviour in the first meiotic prophas e in the mouse chromosomal mutant T(1;13)H/T(1;13)Wa. To preserve cellular shape, primary spermatocytes were encapsulated in a fibrin clot. Convention ally sedimented prophase nuclei served as controls. Axial elements and late ral synaptonemal complex components were subsequently stained by immunofluo rescence and the presence of axial elements at the pachytene stage was high lighted with indirect immunofluorescence against the Atr protein. We compar ed the distribution of Atr signal in the fibrin-embedded spermatocytes with surface-spread preparations and immunohistochemically stained histological sections of seminiferous tubules. Furthermore, fluorescence in situ hybrid isation of the mouse minor satellite DNA was done on fibrin-embedded sperma tocytes. The Atr signal is most conspicuous in fibrin-embedded nuclei on un paired axial elements during pachytene, both for sex chromosomal and for au tosomal segments, and expanding from these elements into the surrounding ch romatin. Both spread and encapsulated zygotene nuclei with extended axial e lement formation proved to be positive for Atr. Mid- to late zygotene nucle i were devoid of 3,3'-diaminodibenzene deposition in the histological secti ons. Highlighting the unpaired axial elements in the small heteromorphic 1( 13)H;1(13)Wa bivalent with an Atr signal enabled meiotic analysis of this b ivalent to be carried out in a three-dimensional context. Thus, proximity o f this bivalent with the sex chromosomes is found more often in three-dimen sional preparations than in spread preparations. Furthermore, the developme nt of the Atr signal over the sex chromosomes as pachytene proceeds helps i n substaging of this long and heterogeneous meiotic phase, in sedimented bu t especially in fibrin-encapsulated nuclei.