Semen Assessments

ProAKAP4 Assessment

A-kinase anchor protein 4 (AKAP4) is a vital sperm protein, functioning in fibrous sheath formation, sperm motility, and fertilisation (specifically capacitation and acrosome reaction). AKAP4 is synthesized from its precursor molecule, proAKAP4, during spermatogenesis [1, 2]. Once formed, AKAP4 is then transported to the developing sperm tail, becoming the most abundant structural element of the fibrous sheath. In mature spermatozoa, it is thought that proAKAP4 then serves as a reservoir for the future production of mature AKAP4 to promote motility when, and if required [2, 3]. Both proAKAP4 and AKAP4 are highly susceptible to adduction by the cytotoxic aldehyde, 4-Hydroxynonenol (4HNE), which is produced during lipid peroxidation. Such adduction deleteriously affects the proteins’ stability and function [4, 5]. Degradation or under expression of proAKAP4 and AKAP4 is associated with decreased sperm quality, motility, and fertility, in human and livestock species [3, 5-8]. For these reasons, proAKAP4 is proving to be a useful biomarker of ejaculate quality in dogs, bulls, rams, stallions, and men [1, 2, 8-10].  

Exclusive to Australia, and in collaboration with SPQI SAS―developers of 4BioDx® and 4MID® product lines―Breed Diagnostics can assess the abundance of proAKAP4 within an ejaculate using the Horse 4MID® proAKAP4 ELISA detection kit. ELISA (enzyme-linked immunosorbent assay) is a highly specific, plate-based technique designed for the detection and quantification of specific proteins and other soluble substances. Spermatozoa are lysed to degrade cell structures and to isolate sperm proteins. The lysis is then aliquoted into the Horse 4MID® microplate wells, that are precoated with proAKAP4 antibodies. Only proAKAP4 will bind to these specific antibodies. Unbound proteins and other cell substances are removed by washing, before a secondary antibody, with conjugated enzyme, is added to each well. These secondary antibodies bind specifically to proAKAP4. Again, unbound antibodies are removed by washing, before a colourogenic substrate is added to each well. The bound conjugated enzyme reacts with this substrate, causing a visible colour change. Where a sample contains a high concentration of proAKAP4, there is a higher level of secondary antibody binding, and therefore a higher degree of colour change (induced by the conjugated enzyme and colourogenic substrate reaction). In contrast, the lower the concentration of proAKAP4, the lower the colour change.

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1. Delehedde, M., Carracedo, S., Selleslagh, M.,Eddarkaoui, S., Amirat-Briand, L., and Sergeant, N. (2020) ProAKAP4 polypeptide as a biomarker of sperm functionality and male fertility disorders. International Journal of Gynecology and Reproductive Sciences. 2 (1): p. 13-19.

2. Sergeant, N., Briand-Amirat, L., Bencharif, D., and Delehedde, M. (2019) The sperm specific protein proAKAP4 as an innovative marker to evaluate sperm quality and fertility. in Journal of Dairy & Veterinary Sciences.  Juniper Publisher. p. 1-6.

3. Jumeau, F., Sigala, J., Dossou-Gbete, F., Frimat, K., Barbotin, A.L., Buee, L., Behal,H., Sergeant, N., and Mitchell, V. (2018) A-kinase anchor protein 4 precursor (pro-AKAP4) in human spermatozoa. Andrology. 6 (6): p. 854-859.

4. 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).

5. Griffin, R.A., Swegen, A., Baker, M., Aitken, R.J., Skerrett-Byrne, D.A., SilvaRodriguez, A., Martín-Cano, F.E., Nixon, B., Peña, F.J., et al. (2020) Mass spectrometry reveals distinct proteomic profiles in high- and low-quality stallion spermatozoa. Reproduction. 160 (5): p. 695-707.

6. Frapsauce, C., Pionneau, C., Bouley, J., Delarouziere, V., Berthaut, I., Ravel, C., Antoine, J.-M., Soubrier, F., and Mandelbaum, J. (2014) Proteomic identification of target proteins in normal but non fertilizing sperm. Fertility and Sterility. 102 (2): p. 372-380.

7. Peddinti, D., Nanduri, B., Kaya, A., Feugang, J.M., Burgess, S.C., and Memili, E. (2008) Comprehensive proteomic analysis of bovine spermatozoa of varying fertility rates and identification of biomarkers associated with fertility. BMC Systems Biology. 2: p. 19.

8. Blommaert, D., Sergeant, N., Delehedde, M., Franck, T., Lejeune, J.-P., and Serteyn, D. (2018) Significant Correlation Between the proAKAP4 Concentration and the total and progressive motility in stallion sperm after thawing. Journal of Equine Veterinary Science. 66: p. 43.

9. Riesco, M.F., Anel-Lopez, L., Neila-Montero, M., Palacin-Martinez, C., Montes-Garrido, R., Alvarez, M., de Paz, P., and Anel, L. (2020) ProAKAP4 as novel molecular marker of sperm quality in ram: An integrative study in fresh, cooled and cryopreserved sperm. Biomolecules. 10 (7).

10. LeCouazer, D., Sergeant, N., Jouy, N., Michaud, S., Loyens, A., Delehedde, M., Amirat-Briand, L., and Bencharif, D. (2019) Expression of proAKAP4 in dog semen as promising marker of sperm quality. Reproduction in Domestic Animals. 54 (S3): p. 73.