ANEUPLOIDY IN LATE-STEP SPERMATIDS OF MICE DETECTED BY 2-CHROMOSOME FLUORESCENCE IN-SITU HYBRIDIZATION

A multicolor procedure employing fluorescence in situ hybridization is described for detecting chromosomal domains and germinal aneuploidy i n late-step spermatids in mice using DNA probes specific for repetitiv e sequences near the centromeres of chromosomes 8 and X. These probes were nick-translated with biotin- or digoxigenin-labeled nucleotides, and were detected with FITC or rhodamine. Probe and hybridization spec ificities were confirmed using metaphase chromosomes from spleen and b one marrow cells as well as from primary and secondary spermatocytes. Late-step spermatids, identified in testicular preparations by their h ooked shape, yielded compact fluorescence domains in similar to 50% an d >99% of cells when hybridized with probes for chromosomes X and 8, r espectively. In a survey of >80,000 late-step spermatids from 8 health y young adult C57BL/6 or B6C3F1 mice, similar to 3/10,000 spermatids h ad fluorescence phenotypes indicative of X-X or 8-8 hyperhaploidy. The se frequencies are consistent with published frequencies of aneuploidy in meiotic metaphase II and first cleavage metaphases of the mouse, p roviding preliminary validation of sperm hybridization for the detecti on of aneuploidy. No significant animal or strain differences were obs erved. In addition, the hyperhaploidy frequencies for murine spermatid s were indistinguishable for those for sperm from healthy men obtained by a similar hybridization procedure. These procedures for detecting aneuploid male gametes are examples of ''bridging biomarkers'' between human and animal studies. They have promising applications for invest igations of the genetic, reproductive, and toxicological factors leadi ng to abnormal reproductive outcomes of paternal origin, (C) 1995 Wile y-Liss, Inc.