Distribution of Atr protein in primary spermatocytes of a mouse chromosomal mutant: a comparison of preparation techniques |
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Authors: | Esther B Baart Dirk G de Rooij Kathleen S Keegan Peter de Boer |
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Institution: | (1) Laboratory of Genetics, Wageningen Institute of Animal Sciences, 6709 PG Wageningen, The Netherlands, NL;(2) Department of Cell Biology, University Medical Center Utrecht, 3584 CX Utrecht, The Netherlands, NL;(3) ICOS Corporation, Bothell, WA 98021, USA, US |
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Abstract: | In this study, we examined the suitability of a three dimensional preparation technique for studying chromosome behaviour
in the first meiotic prophase 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. Conventionally sedimented prophase nuclei served as controls. Axial elements and lateral
synaptonemal complex components were subsequently stained by immunofluorescence and the presence of axial elements at the
pachytene stage was highlighted with indirect immunofluorescence against the Atr protein. We compared 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 hybridisation of the mouse minor satellite DNA was done
on fibrin-embedded spermatocytes. The Atr signal is most conspicuous in fibrin-embedded nuclei on unpaired axial elements
during pachytene, both for sex chromosomal and for autosomal segments, and expanding from these elements into the surrounding
chromatin. Both spread and encapsulated zygotene nuclei with extended axial element formation proved to be positive for Atr.
Mid- to late zygotene nuclei were devoid of 3,3′-diaminodibenzene deposition in the histological sections. Highlighting the
unpaired axial elements in the small heteromorphic 113H;113Wa bivalent with an Atr signal enabled meiotic analysis of this bivalent to be carried out in a three-dimensional context.
Thus, proximity of this bivalent with the sex chromosomes is found more often in three-dimensional preparations than in spread
preparations. Furthermore, the development of the Atr signal over the sex chromosomes as pachytene proceeds helps in substaging
of this long and heterogeneous meiotic phase, in sedimented but especially in fibrin-encapsulated nuclei.
Received: 22 September 1999; in revised form: 20 December 1999 / Accepted: 21 December 1999 |
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