Semen Assessments
When a cell is experiencing oxidative stress, lipids in the plasma membrane become oxidized and degraded; a process known as lipid peroxidation. They cytotoxic aldehyde, 4-hydroxynonenol (4HNE), is produced as a byproduct of lipid peroxidation. 4HNE is highly reactive, disrupting the function of many vital sperm proteins [1, 2], and directly suppressing sperm motility and fertilizing potential [3, 4]. Therefore, assessment of 4HNE concentration provides an indirect measure of the extent of lipid peroxidation.
Spermatozoa are firstly incubated with a vitality stain (Live/Dead™; LD), and a sequential antibody incubation is then performed, whereby the primary antibody specifically binds to 4HNE (the antigen), and the secondary antibody, with conjugated fluorophore, binds to the primary antibody. Any unbound antibodies are removed by washing. The use of specific fluorescent antibodies enables us to assess the extent of lipid peroxidation within an ejaculate using flow cytometry. The reported populations are:
The process and effects of lipid peroxidation are discussed by Dutta [5], and in greater detail by Nowicka-Bauer and Nixon [6].
1. Bromfield, E.G., Aitken, R.J., McLaughlin, E.A., and Nixon, B. (2017) Proteolytic degradation of heat shock protein A2 occurs in response to oxidative stress in male germ cells of the mouse. Molecular Human Reproduction. 23(2): p. 91-105.
2. Nixon, B., Bernstein, I.R., Cafe, S.L., Delehedde, M., Sergeant, N., Anderson, A.L., Trigg, N.A., Eamens, A.L., Lord, T., et al. (2019) A Kinase Anchor Protein 4 Is Vulnerable to Oxidative Adduction in Male Germ Cells. Frontiers in Cell and Developmental Biology. 7(319).
3. Gibb, Z., Lambourne, S.R., Curry, B.J., Hall, S.E., and Aitken, R.J. (2016) Aldehyde Dehydrogenase Plays a Pivotal Role in the Maintenance of Stallion Sperm Motility. Biology of Reproduction. 94(6): p. 133.
4. Hall, S.E., Aitken, R.J., Nixon, B., Smith, N.D., and Gibb, Z. (2017) Electrophilic aldehyde products of lipid peroxidation selectively adduct to heat shock protein 90 and arylsulfatase A in stallion spermatozoa. Biology of Reproduction. 96(1): p. 107-121.
5. Dutta, S., Majzoub, A., and Agarwal, A. (2019) Oxidative stress and sperm function: A systematic review on evaluation and management. Arab Journal of Urology. 17(2): p. 87-97.
6. Nowicka-Bauer, K. and Nixon, B. (2020) Molecular Changes Induced by Oxidative Stress that Impair Human Sperm Motility. Antioxidants. 9(2): p. 134.