COMPARISON OF DETERGENT-SOLUBILIZED MEMBRANE AND SOLUBLE-PROTEINS FROM FLOW CYTOMETRICALLY SORTED X-CHROMOSOME AND Y-CHROMOSOME BEARING PORCINE SPERMATOZOA BY HIGH-RESOLUTION 2-D ELECTROPHORESIS

The only known and measurable difference between X- and Y-chromosome b earing spermatozoa is the small difference in their DNA content. The X sperm in the human carry 2.8% more DNA than the Y sperm, while in dom estic livestock this difference ranges from 3.0 to 4.2%. The only succ essful sperm separation method, flow cytometric sorting, is based on t his difference in DNA content. Using this technique, X and Y sperm pop ulations with purities greater than 90% can be obtained. The number of spermatozoa that can be sorted in a given time period, however, is to o low for application of this technique in routine artificial insemina tion. Therefore, the search for a marker other than DNA to differentia te between X and Y sperm remains of interest in order to develop a met hod for large scale X and Y sperm separation. The aim of the present s tudy was to investigate whether porcine X and Y sperm contain some dif ference in their plasma membrane proteins. The flow cytometric sorting of sperm enabled a direct comparison of the proteins of the X and Y s perm populations. High resolution two-dimensional (2-D) electrophoresi s was used; however, adaptations were needed to enable its use for ana lysis of proteins of flow cytometrically sorted sperm, both in the sor ting procedure, membrane protein solubilization, and in the 2-D electr ophoresis. Up to 1000 protein spots per gel could be detected and quan tified. Comparison of the 2-D protein patterns revealed differences in protein spots between sperm of two individual boars. However, no diff erences in protein spots between the X and Y sperm fractions were foun d. These results provide additional support for the view that X- and Y -chromosome bearing spermatozoa are phenotypically identical, and cast doubt on the likelihood that a surface marker can provide a base for X and Y sperm separation. (C) 1996 Wiley-Liss, Inc.