Morphological detection of X- and Y-chromosomes in smears and paraffin-embedded tissues using a non-isotopic in situ hybridization technique (NISH)

Abstract

Pharyngeal smears and paraffin-embedded tissue specimens (skeletal muscle, kidney) obtained from 10 male and 10 female individuals were evaluated using non-isotopic in situ hybridization (NISH) with commercial X-and Y-specific biotinylated probes which recognize the pericentromeric regions DXZ1 and DYZ1/DYZ3 of the X-and Y-chromosome, respectively. The results provide evidence that the morphological sex determination of a single cell can be performed by critical application of this staining method leading to one nuclear signal in "male" cells using the Y-specific probe whereas "female" cells are negative. In situ hybridization of "female" tissues with an X-specific probe results regularly in 2 signals whereas "male" cells show only one spot in the nucleus.

Optimization of Non-isotopic in situ Hybridization: Detection of the Y Chromosome in Paraformaldehyde-Fixed, Wax-Embedded Cat Retina

“…Proteinase K and pepsin are popular proteases used to increase availability of probe binding sites (Burns et al, 1987 ;Katsumata and Lo, 1988 ;Dekken et al, 1992 ;Betz et al, 1994 ;Nagler et al, 1994). Treatment with proteinase K failed to rescue probe binding sites in our tissue.…”

“…Recently, the use of microwaves to retrieve masked epitopes has enhanced significantly both immunohistochemical and ISH signals (Shi, Key and Kalra, 1991 ;Hsu, Peng and Shun, 1991 ;McMahon and McQuaid, 1996 ;Portiansky and Gimeno, 1996). However, many investigators experience technical difficulty in performing ISH on routinely processed tissue (Scholefied et al, 1990 ;Arnoldus et al, 1991 ;Betz et al, 1994). We carried out a systematic study to seek the optimal pretreatment conditions for ISH detection of the Y chromosome in different nuclear layers of the retina and RPE in paraformaldehyde-fixed, DGD embedded cat tissue.…”

Gender verification in sports by PCR amplification of SRY and DYZ1 Y chromosome specific sequences: presence of DYZ1 repeat in female athletes.

“…In our previous studies, amplification of DYZ1 specific sequences was detected in 100% of the control male samples (89/89), and in none of 1629 female samples. However, when the female competitors from the Barcelona Olympic Games were analysed with this assay, 11 samples rendered a positive result (11/2406; 0.46%). In a second screening, SRY and DYZ1 specific sequences were amplified from purified DNA from these 11 samples, using primers 51-S2 and D1-D2.…”