Phytohaemagglutinin as a molecular probe to study the membrane constituents of human X- and Y-bearing spermatozoa.

Phytohaemagglutinin (PHA) was employed to study the constituents of the cell membrane in X- and Y-bearing human spermatozoa. The presence of certain receptors for PHA was revealed in the equatorial region of about 50% of the spermatozoa. To determine the association of the equatorial staining by PHA with one of the subpopulations of spermatozoa a double staining technique was developed. In this method, the membrane was first stained with PHA employing the indirect immunofluorescence technique and then followed by the conventional quinacrine staining method to demonstrate the F-body. It was shown that spermatozoa which had receptors in the equatorial region for PHA were the ones which exhibited in the F-body. Staining of the membrane with PHA is a better method for the identification of human Y spermatozoa since it does not have the drawbacks of quinacrine staining.

A simple and reproducible method for separating Y-bearing spermatozoa from human semen.

PIP: A simple and reproducible method for separating Y-bearing spermatozoa from human semen is reported. Semen samples with 60-80 million count and 50% motility were obtained from healthy human donors. Ficoll-400 and sodium metrizoate density gradient were used for separation. Quinacrine hydrochloride was used for staining, and Olympus fluorescent microscopy was used for the detection of F body present in the spermatozoa bearing Y-chromosomes. The data indicate that the percentage of Y-bearing spermatozoa in normal human semen is about 5-6% less than what is expected. The observations agree with those of Barlow and Vosa that with the optical attenuation coefficient being sufficiently great, the chromosomes lying deep in the head may go undetected. A mean value of 45.22 was obtained for Y-bearing spermatozoa and the sediment contained about 62.9 Y-bearing spermatozoa. The results thus suggest that it is possible to separate Y-bearing spermatozoa from semen using Ficoll-socium metrizoate density gradient.^ieng

Use of dithiothreitol for improved visibility of the F body in human Y-bearing spermatozoa.

Dithiothreitol was used to decondense the nuclear chromatin before staining human spermatozoa with quinacrine for the F body present in Y-bearing spermatozoa. In unseparated fractions and those enriched in X- or Y-bearing spermatozoa, the number of spermatozoa with F bodies increased by 2-9% after dithiothreitol treatment.