Patricia Emmerich

Double in situ hybridization in combination with digital image analysis: a new approach to study interphase chromosome topography

Double in situ hybridization with mercurated and biotinylated chromosome specific DNA probes in combination with digital image analysis provides a new approach to compare the distribution of homologous and nonhomologous chromosome targets within individual interphase nuclei. Here we have used two DNA probes representing tandemly repeated sequences specific for the constitutive heterochromatin of the human chromosomes 1 and 15, respectively, and studied the relative arrangements of these chromosome targets in interphase nuclei of human lymphocytes, amniotic fluid cells, and fibroblasts, cultivated in vitro. We have developed a 2D-image analysis approach which allows the rapid evaluation of large numbers of interphase nuclei. Models to test for a random versus nonrandom distribution of chromosome segments are discussed taking into account the three-dimensional origin of the evaluated 2D-distribution. In all three human diploid cell types the measurements of target-target and target-center distances in the 2D-nuclear image revealed that the labeled segments of the two chromosomes 15 were distributed both significantly closer to each other and closer to the center of the nuclear image than the labeled chromosome 1 segments. This result can be explained by the association of nucleolus organizer regions on the short arm of chromosome 15 with nucleoli located more centrally in these nuclei and does not provide evidence for a homologous association per se. In contrast, evaluation of the interphase positioning of the two chromosome 1 segments fits the random expectation in amniotic fluid and fibroblast cells, while in experiments using lymphocytes a slight excess of larger distances between these homologous targets was occasionally observed. 2D-distances between the labeled chromosome 1 and 15 segments showed a large variability in their relative positioning. In conclusion our data do not support the idea of a strict and permanent association of these homologous and nonhomologous targets in the cell types studied so far.

Chimeric human and mouse spheroids

SummaryWe investigated structures resembling embryoid bodies (EBs), grown intraperitoneally in nude mice after the injection of xenografted human teratocarcinoma cells. Following in situ hybridization of paraffin sections containing these EB-like structures with either human or mouse total genomic DNA, two species-specific types of cell nuclei were localized. Tumor cells of human origin were found centrally but flattened normal mouse cells formed an outer coat. Thus these spheric structures are of bispecies origin and do not meet the definition of EBs. For a clear distinction from EBs and spheroids, we termed these structures chimeric spheroids.

Characterization of Precancerous and Neoplastic Human Testicular Germ Cells

Testicular germ cell tumors are in most cases preceded by an intratubular noninvasive form of neoplasia, known as carcinoma in situ (CIS, Skakkebaek and Berthelsen 1978). These atypical germ cells resemble gonocytes or spermatogonia (Nuesch-Bachmann and Hedinger 1977), but also show nuclear atypia indicative of malignant transformation. Atypical intratubular cells have been known to precede overt tumors for up to 16 years (Bannwart et al. 1988). However, they do not have invasive properties and the attempts to grow them as xenografts in nude mice have been unsuccessful (Walt and Stevens unpublished results). The transition of CIS to invasive carcinoma therefore remains poorly understood. In an effort to understand better the pathobiology of testicular neoplasias and their precancerous precursors, we have studied the expression of certain biochemical tumor markers (Walt et al. 1986) and of alkaline phosphatase isozymes (Hofmann et al. 1989), the activity of nucleolus organizer regions (NORs) (Delozier-Blanchet et al. 1986a), and the presence of a number of specific chromosomal regions by in situ hybridization on paraffin sections (Emmerich et al. 1989). For these investigations we have used xenografts and their related cell lines (DelozierBlanchet et al. 1986b; Emmerich et al. 1989; Hofmann et al. 1989; Walt et al. 1986) as well as primary tumors and primary precancerous and ejaculated germ cells (Jauch et al. 1989; Walt et al. 1989). We hope that this summary of data will allow the development of new strategies for further research in this area.

Computer aided 3D localization of Chromosome 1 within nuclei of human sperm cells

Conventional cellular and histological analyses are generally based on planar images. New preparation techniques in the field of immunohistochemistry and in situ hybridization demand a more sophisticated analytical approach. Our method to detect irregularities in the number of chromosomes present in interphase nuclei of normal and neoplastic cells and to characterize aberrations in the structure of these chromosomes makes use of a combination of confocal laser scanning microscopy (CLSM) and 3-D imaging. In this paper we describe a computer-assisted setup designed to localize in situ hybridized chromosome 1 of human sperm cells. In order to synthesize a 3-D image of the structures of interest, the stack of slice images generated by the CLSM is processed appropriately with the aid of a parallel distributed computer architecture based on transputers. The resulting images or particular aspects thereof are visualized on a high resolution display and related to new information in reproductive biology.