Oral Virtual Presentation (Virtual only) ESA-SRB-ANZBMS 2021

Shifting the paradigm of sperm maturation; global proteomic profiling of epididymal sperm (#117)

David A Skerrett-Byrne 1 2 , Amanda L Anderson 1 2 , Elizabeth G Bromfield 1 2 3 , Ilana R Berntstein 1 2 , Jess E Mulhall 1 2 , John E Schjenken 1 2 , Sean J Humphrey 4 5 , Brett Nixon 1 2
  1. Priority Research Centre for Reproductive Science, School of Environmental and Life Sciences College of Engineering, Science and Environment, The University of Newcastle, Newcastle, New South Wales, Australia
  2. Pregnancy and Reproduction Program, Hunter Medical Research Institute, Newcastle, New South Wales, Australia
  3. Faculty of Veterinary Medicine, Utrecht University, Utrecht , The Netherlands
  4. School of Life and Environmental Sciences, The University of Sydney, Sydney, New South Wales, Australia
  5. The Charles Perkins Centre, The University of Sydney, Sydney, New South Wales, Australia

Prior to engaging in fertilisation, mammalian spermatozoa must first complete an arduous journey of functional maturation as they transit the epididymis. A distinctive feature of this process is that it occurs in the absence of de novo gene transcription and protein translation and is thus reliant on a substantive remodelling of the intrinsic sperm proteomic architecture, the scale of which is yet to be fully understood. Here, we have sought to define the extent of proteomic changes associated with the maturation of mouse spermatozoa using label-free quantitative mass spectrometry; reporting an unprecedented depth of coverage encompassing >6,000 proteins (Fig. A). Contrary to the long-held belief that epididymal maturation is primarily driven by the uptake of additional proteins, our data demonstrates that sperm shed over 56% of their proteins during this process, producing a refined fertilisation-competent cell (Fig. B; 9% gain). Complementing these extensive changes, the abundance of an additional 889 proteins was significantly altered as sperm transited the epididymis (≥2-fold change, p≤0.05). Furthermore, counterintuitive to the notion of gaining proteins to gain function, the reduced complexity of the sperm proteome aligned with the putative activation of key cellular functions including sperm ‘motility’ and ‘capacitation’. In accounting for how these proteomic changes influence sperm function, we demonstrate that RHOA, a small GTPase, is acquired by maturing spermatozoa (2-fold increase), with a concomitant downregulation or complete loss of RHOA repressing proteins, including ARHGAP18, ARHGDIB, GDI-1, in mature sperm. To investigate the function of RHOA we pharmacologically inhibited its activity, resulting in a compromise to the ability of mature spermatozoa to undergo an acrosome reaction (Fig. C; ~40% reduction). These data contribute a new understanding of the mechanisms that underpin the transformation of sperm into functionally competent cells and provide a platform to identify proteins that equip sperm for fertilisation.

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