Aneuploidy in late‐step spermatids of mice detected by two‐chromosome fluorescence in situ hybridization

Abstract

A multicolor procedure employing fluorescence in situ hybridization is described for detecting chromosomal domains and germinal aneuploidy in late-step spermatids in mice using DNA probes specific for repetitive 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 specificities were confirmed using metaphase chromosomes from spleen and bone marrow cells as well as from primary and secondary spermatocytes. Late-step spermatids, identified in testicular preparations by their hooked shape, yielded compact fluorescence domains in approximately 50% and > 99% of cells when hybridized with probes for chromosomes X and 8, respectively. In a survey of > 80,000 late-step spermatids from 8 healthy young adult C57BL/6 or B6C3F1 mice, approximately 3/10,000 spermatids had fluorescence phenotypes indicative of X-X or 8-8 hyperhaploidy. These frequencies are consistent with published frequencies of aneuploidy in meiotic metaphase II and first cleavage metaphases of the mouse, providing preliminary validation of sperm hybridization for the detection of aneuploidy. No significant animal or strain differences were observed. In addition, the hyperhaploidy frequencies for murine spermatids 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 investigations of the genetic, reproductive, and toxicological factors leading to abnormal reproductive outcomes of paternal origin.

Micronuclei induced in round spermatids of mice after stem-cell treatment with chloral hydrate: Evaluations with centromeric DNA probes and kinetochore antibodies

“…la) and SMN using a DAPI filter set (Zeiss filter 487901) as described by Wyrobek et al [1995]. Based on the presence of the Xchromosome probe (green label) and pan-centromeric probe (red label), SMN were classified into one of four phenotypes: (1) DAF' I-and pancentromeric-positive ( …”

“…Hybridizations were carried out as described by Wyrobek et al [I9951 with the following modifications: cells were pretreated for 5 min with 1% RNase (Boehringer Mannheim, Indianapolis, IN) in 2XSSC and washed for 5 min in PN buffer (NaHPO,, NaH2P04, and Nonidet P-40 detergent) prior to denaturation; following hybridization, slides were washed three times in washing solution (50% formamide, 2XSSC) and two times in 2XSSC at 45°C for 10 min each, and then twice in room temperature PN buffer for 10 min each. Digoxigenin-labeled pan-centromeric probe and biotinylated X probe were detected with anti-digoxigenin antibody conjugated with rhodamine and with Avidin D conjugated to FITC, respectively, as described by Wyrobek et al [1995]. DAPI (0.5 pg/&, Sigma Chemical Co., St. Louis, MO) in antifade (Vectashield Mounting Medium, Vector Labs, Burlingame, CA) was used as a counterstain.…”

“…Two DNA probes were used for hybridization: DXWas-70, specific for mouse chromosome X, a gift from Dr. Christine Disteche [Disteche et al, 19851, and mouse Cot-1-DNA, specific to centromeric DNA of all mouse chromosomes except chromosome Y (Gibco BRL, Gaithersburg, MD). Transformation, growth, and isolation of the plasmid harboring the X probe, were as described by Wyrobek et al [1995]. Nick translation (Nick Translation System, Gibco BRL) was used to label the X-chromosome and the pancentromeric probes with biotin-14-dATP (Gibco BRL) and digoxigenin-1 I-dUTP (Boehringer Mannheim, Indianapolis, IN), respectively.…”

The use of fluorescent in situ hybridization in male infertility

“…In 1995, a mouse testicular sperm aneuploidy assay was developed to detect aneuploidy in sex and autosomal chromosomes in mouse spermatozoa [Wyrobek et al 1995] and later expanded to assess epididymal sperm [Lowe et al 1996]. Eventually, the same group developed a similar multicolor sperm-FISH assay in rats ].…”

Hybridization-based karyotyping of mouse chromosomes: hybridization-bands

“…Hyb-banding may be useful as a preliminary screening technique to identify quickly those chromosomes involved in rearrangements, prior to the application of traditional banding methods or chromosome-specific fluorescent painting probes. Aneuploidies are already being assessed by means of hybridization probes (Wyrobek et al, 1995); Hyb-bands can be applied in studies of aneuploidy and mutagenesis to determine quickly which chromosomes are affected, as illustrated in Fig. 3.…”