Semen Assessments
Chromatin is a complex combination of DNA and nuclear proteins that make up chromosomes. During early spermatogenesis, proteins known as histones act as spools that DNA winds around enabling compaction. As the cell continues to mature, histones are removed and replaced by protamines, which further compact the DNA into a tightly bound toroid. The histone-to-protamine transition enable sperm head condensation, DNA stabilisation and importantly, protects DNA from oxidative stress. Any disruption to this process has profound effects on DNA integrity, future fertilisation capacity and embryonic development [1-3]. The sperm chromatin structure assay (SCSA) retrospectively assesses the DNA compaction process, thus providing an indirect measure of the susceptibility of DNA to damage.
Snap-frozen spermatozoa are thawed, and incubated with an acid detergent solution that denatures DNA at sites of DNA strand breaks. Spermatozoa are subsequently stained with acridine orange and assessed using flow cytometry. Double stranded DNA (intact DNA) fluoresces green, while single stranded DNA (denatured due to pre-existing DNA strand breaks and poor chromatin formation) fluoresces red. SCSA assessments have been shown to be predictive of fertility in man [4], bull [5, 6], boar [7] and stallions [8, 9], among other species.
1. Szczygiel, M.A. and Ward, W.S. (2002) Combination of Dithiothreitol and Detergent Treatment of Spermatozoa Causes Paternal Chromosomal Damage1. Biology of Reproduction. 67(5): p. 1532-1537.
2. Ward, W.S. and Coffey, D.S. (1991) DNA packaging and organization in mammalian spermatozoa: comparison with somatic cells. Biology of Reproduction. 44(4): p. 569-74.
3. Zhang, X., San Gabriel, M., and Zini, A. (2006) Sperm nuclear histone to protamine ratio in fertile and infertile men: evidence of heterogeneous subpopulations of spermatozoa in the ejaculate. J Androl. 27(3): p. 414-20.
4. Giwercman, A., Lindstedt, L., Larsson, M., Bungum, M., Spano, M., Levine, R.J., and Rylander, L. (2010) Sperm chromatin structure assay as an independent predictor of fertility in vivo: a case–control study. International Journal of Andrology. 33(1): p. e221-e227.
5. Bochenek, M., Smorąg, Z., and Pilch, J. (2001) Sperm chromatin structure assay of bulls qualified for artificial insemination. Theriogenology. 56(4): p. 557-567.
6. Waterhouse, K.E., Haugan, T., Kommisrud, E., Tverdal, A., Flatberg, G., Farstad, W., Evenson, D.P., and De Angelis, P.M. (2006) Sperm DNA damage is related to field fertility of semen from young Norwegian Red bulls. Reproduction, Fertility and Development. 18(7): p. 781-788.
7. Didion, B.A., Kasperson, K.M., Wixon, R.L., and Evenson, D.P. (2009) Boar Fertility and Sperm Chromatin Structure Status: A Retrospective Report. Journal of Andrology. 30(6): p. 655-660.
8. Love, C.C. and Kenney, R.M. (1999) Scrotal heat stress induces altered sperm chromatin structure associated with a decrease in protamine disulfide bonding in the stallion. Biology of Reproduction. 60(3): p. 615-20.
9. Love, C.C. (2005) The sperm chromatin structure assay: A review of clinical applications. Animal Reproduction Science. 89(1): p. 39-45.