Analysis of cell ploidy in histological sections of mouse tissues by DNA-DNA in situ hybridization with digoxigenin-labelled probes

Abstract

DNA-DNA in situ hybridization, with two digoxigenin-labelled, chromosome-specific DNA probes, was used to determine the number of copies of a given chromosome in interphase nuclei and so identify putatively polyploid nuclei in histological sections of several mouse tissues. One hybridization site per diploid genome was expected for tissues with hemizygous markers: male mice hybridized with a Y chromosome probe (pY353/B) or hemizygous transgenic mice hybridized with a beta-globin probe (pM beta delta 2). Nuclei with more than one hybridization site were considered putative polyploids. Three groups of experiments were undertaken: (1) evaluation of the method, using mouse liver sections; (2) studies of tissues already known to contain polyploid nuclei, and (3) studies that resulted in the discovery that the mouse ovary contains polyploid nuclei. First, control studies showed that the ability to detect the target DNA sequences was affected by section thickness. Studies of nuclear ploidy in the developing mouse liver revealed a pattern similar to that established by previous studies using DNA content as a criterion for ploidy. At birth, only about 5% of the liver nuclei were polyploid; this increased to 10-15% by 10-20 days and was followed by a sharp increase in the frequency of tetraploid nuclei between 20 and 40 days (to about 35%) and a more gradual increase in higher order polyploid nuclei. Secondly, this technique was used to confirm that polyploid (mostly tetraploid) nuclei were present in the bladder epithelium, heart, uterine decidua and placental trophoblast. Higher order polyploidy was seen in large bone marrow cells (megakaryocytes) but not in the even larger trophoblast giant cells of the placenta, thus confirming previous claims that these cells are polytene rather than polyploid. Thirdly, putatively tetraploid nuclei were found in the ovarian follicle and corpus luteum. As far as we are aware, this is the first time polyploid nuclei have been reported for the mouse ovary.

Mouse Strain Susceptibility to Diethylnitrosamine Induced Hepatocarcinogenesis Is Cell Autonomous Whereas Sex‐susceptibility Is Due to the Micro‐environment: Analysis with C3H ↔ BALB/c Sexually Chimeric Mice

“…Thus, the Y353/B Y chromosome-specific marker 18) was chosen, because 30 copies of the cognate and 250 copies of related sequences exist along the length of the Y chromosome. 29) This chromosomal marker has been used as a hemizygous marker for liver sections 30) and for transplanted hepatocytes 31) with strong reproducibility. The Y chromosome-specific marker can visualize XY cells directly even when the chimerism ratio is extreme so that Sry SSLP analysis is impossible.…”

Analysis of mouse eye development with chimeras and mosaics

“…However, transgenic markers are now more commonly used. These include the highly reiterated Tg(Hbb-b1)83Clo transgene, which can be detected in histological sections by DNA in situ hybridisation (Keighren and West, 1993;Lo, 1986), and transgenes that express ubiquitously either lac Z, such as Gt(ROSA)26Sor (ROSA26) (Friedrich and Soriano, 1991), or green fluorescent protein (GFP) (Hadjantonakis et al, 1998a;Pratt et al, 2000).…”

Three‐dimensional reconstruction of tetraploid↔diploid chimaeric mouse blastocysts

“…Serial sections were cut at 7 µm and in situ hybridisation was carried out, using the digoxygenin-labelled probe pMβδ2 (Lo et al 1987) and a diaminobenzidine endpoint (Keighren & West, 1993), to detect the β-globin sequences. In this way, the hemizygous diploid (Tg\-) or tetraploid cells (Tg\Tg\-\-), bearing 1 or 2 copies of the β-globin transgene respectively could be distinguished from cells from the other contributing embryo.…”