176 Characterisation of proAKAP4 expression, metabolism, and localisation in Equus asinus spermatozoa
M. Dordas-Perpinya A B , N. Sergeant C D , I. Yanez-Ortiz A , J. Catalan A , M. Delehedde D , J. Bruyas B , L. Briand-Amirat B and J. Miro AA Servei de Reproducció Equina, Departament de Medicina i Cirurgia Animals, Universitat Autónoma de Barcelona, Bellaterra, Spain
B Oniris, Nantes, Loire-Atlantic, France
C University of Lille, Inserm UMRS1172, CHRU Lille, Lille, France
D SPQI, 4BioDx, Lille, France
Reproduction, Fertility and Development 35(2) 215-216 https://doi.org/10.1071/RDv35n2Ab176
Published: 5 December 2022
© 2023 The Author(s) (or their employer(s)). Published by CSIRO Publishing on behalf of the IETS
ProAKAP4 is the AKAP4 precursor that belongs to the A-kinase anchoring protein (AKAP) family and is described as a sperm protein marker related to spermatozoa maturity, long-lasting motility, and sperm functionality. The aim of this work was to study, for the first time, proAKAP4 expression and metabolism in donkey semen and compared to stallion semen. Ejaculates from five Catalan donkeys and five stallions were collected during several weeks using a Hannover artificial vagina in our reproduction service from UAB. After dilution in extender, they were then packed in straws of 100 million of spermatozoa and frozen following the commercial protocol of the service. We then analysed proAKAP4 and AKAP4 expressions in post-thaw conditions. In donkeys, as in stallions, both polypeptides were observed by Western blotting with the detection of a band at 100 kDa for proAKAP4 (clone6F12 antibody) and at 80 kDa for AKAP4 (clone 7F10 antibody). The cleavage process of the proAKAP4 as previously described for other mammals is then preserved in Catalan donkey sperm and released both the mature AKAP4 and the small prodomain of around 20 kDa. A 1:1 ratio between polypeptide was quantified using β-actin and reflected a conserved proAKAP4 metabolism in Catalan donkeys. By immunofluorescence, we observed that both proAKAP4 and AKAP4 were decorating the principal piece of the flagellum of donkey spermatozoa. Interestingly, not all donkey spermatozoa were labelled, highlighting a differential expression and/or metabolism in individual post-thaw spermatozoa from donkey and stallion spermatozoa. The amount of proAKAP4 in post-thawed donkey spermatozoa was then quantified by ELISA, and interindividual variations in proAKAP4 concentrations were observed in donkeys with concentration levels comparable to other mammals. ProAKAP4 concentrations in ng/10 million of spermatozoa are indicative of the full-length AKAP4 and reflected the disposable amount of AKAP4 available for sperm functionality in post-thawed Catalan donkey semen. In conclusion, for the first time, both AKAP4 and its precursor, proAKAP4, were detected in frozen donkey sperm and may represent a reliable marker to follow-up and further investigate with other sperm parameters to assess more closely sperm functionality and fertility outcomes in Catalan donkeys.
