Abstract
Mammalian sperm are transcriptionally and translationally inactive. To meet changing needs in the epididymis and female tract, they rely heavily on post-translational modifications and protein acquisition/degradation. Membrane rafts are sterol and sphingolipid-enriched micro-domains that organize and regulate various pathways. Rafts have significance in sperm by transducing the stimulus of sterol efflux into changes in intracellular signaling that confer fertilization competence. We recently characterized three biochemically distinct sub-types of sperm rafts, and now present profiles for proteins targeting to and associating with these sub-types, along with a fraction largely comprised of "non-raft" domains. Proteomics analysis using a gel-based LC-MS/MS approach identified 190 strictly validated proteins in the raft sub-types. Interestingly, many of these are known to be expressed in the epididymis, where sperm membrane composition matures. To investigate potential roles for rafts in epididymal protein acquisition, we compared the expression and localization of two different sterol-interacting proteins, apolipoprotein-A1 (apoA1) and prominin-1 (prom1) in sperm from different zones. We found that apoA1 was gradually added to the plasma membrane overlying the acrosome, whereas prom1 was not, suggesting different mechanisms for raft protein acquisition. Our results define raft-associating proteins, demonstrate functional similarities and differences among raft sub-types, and provide insights into raft-mediated epididymal protein acquisition.
Highlights
After leaving the testis, mammalian sperm must undergo two distinct maturational processes in order to become fertilization competent
We showed that this non-detergent based method separates membrane raft sub-types with high purity and reproducibility by means of their buoyancy [9]
To confirm the quality and identity of fractions obtained from the linear density gradient, we analyzed them using refractometry to ensure that the collected fractions represented the same sucrose densities as those we had described (Fig. 2B; note that we performed the fractionation of the sperm membranes 10 times, with very high reproducibility in terms of the biochemical characterization and the refractive indices between replicates [9])
Summary
Mammalian sperm must undergo two distinct maturational processes in order to become fertilization competent. After storage in the cauda and ejaculation, sperm are still unable to fertilize an oocyte until they mature in the female tract in response to external stimuli in the process of “capacitation” [3] This second, functional maturation is associated with multiple physiological events taking place at the level of the plasma membrane, including a requirement for sterol efflux. Because true rafts are only a subset of what might partition with DRM (as they can artifactually cause disparate molecules to coalesce), and because resistance to solubilization is detergent-dependent and doesn’t correspond with known physiological entities, identification of proteins that partition to DRM will at best yield candidates for targeting to or associating with, true rafts [7] Despite this limitation, DRM from sperm have shown interesting capabilities, such as possessing components that bind to the zona pellucida [10,11], making the further characterization of rafts of great interest
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