Flow sorting of X and Y chromosome-bearing spermatozoa into two populations
Article first published online: 17 FEB 2005
Copyright © 1987 Alan R. Liss, Inc.
Volume 16, Issue 1, pages 1–9, January 1987
How to Cite
Johnson, L. A., Flook, J. P., Look, M. V. and Pinkel, D. (1987), Flow sorting of X and Y chromosome-bearing spermatozoa into two populations. Gamete Res., 16: 1–9. doi: 10.1002/mrd.1120160102
- Issue published online: 17 FEB 2005
- Article first published online: 17 FEB 2005
- Manuscript Accepted: 11 JUN 1986
- Manuscript Received: 27 MAR 1986
- flow cytometry;
- sperm separation;
- fluorescent stain
The only established difference on which to base the separation of X and Y chromosome-bearing spermatozoa is chromosomal constitution. This difference is quantifiable both from chromosome morphology (karyotype) and from DNA content. Flow cytometric techniques were used to measure relative DNA content of the X and Y populations and to flow-sort spermatozoa from Chinchilla laniger. Epididymal spermatozoa were recovered in PBS, fixed in 80% ethanol, treated with papain and dithioerythritol, and stained for DNA with Hoechst 33342. Sperm nuclei were analyzed and sorted on an EPICS V flow cytometer/cell sorter, modified specifically for spermatozoa. Two clearly resolved peaks (coefficient of variation < 1.5%) with approximately 7.5% difference in DNA content between X and Y chromosome-bearing spermatozoa were evident. Sperm nuclei were sorted from a portion of the X and Y peaks at a rate of 55 nuclei/sec for each population. Purities of individual X and Y populations averaged 95% as determined by reanalysis of the sorted populations. Successful sorting of Chinchilla X and Y chromosome-bearing spermatozoa into separate populations may aid in the identification of a biochemical marker that could be used to discriminate between the two sperm populations and lead to a practical procedure for sexing spermatozoa.