Dynamic sperm proteome remodeling during epididymal maturation in a marsupial, Macropus giganteus†.

While assisted reproductive technologies (ARTs) are widely used in domestic animals, successful implementation of ARTs to conserve wildlife species remains challenging. In macropods, crucial aspects of fundamental reproductive biology, including changes induced by epididymal maturation, remain unknown, limiting the development of ARTs. In this context, we performed a proteomic analysis of spermatozoa from the caput, corpus, and cauda epididymis of Eastern Grey Kangaroos (n = 6) to profile changes over epididymal maturation. Samples prepared by filter-aided sample preparation digestion were analyzed by liquid chromatography tandem mass spectrometry with sequential window acquisition of all theoretical acquisition. A total of 4304 proteins were identified, with significant overlap across epididymal regions. Highly abundant proteins in common across caput, corpus, and cauda spermatozoa had strong enrichment for tubulins and included four histone proteins. The most significant proteomic remodeling was observed in the corpus to cauda transition, late in epididymal transit (728 differentially abundant proteins). Overall proteomic changes across epididymal maturation (1131 differentially abundant proteins) suggested a loss of sperm glycosidases and an increase in flagellar proteins, including tubulins and dyneins. These findings serve to highlight both consistencies with eutherian sperm epididymal maturation (e.g. bias toward protein loss over transit, transfer of proteins via extracellular vesicles) and elements which are likely unique to marsupials (e.g. reduced chromatin stability, potential use of β-oxidation as a major metabolic pathway). This critical information can now be leveraged to further develop ARTs in marsupials.