Characterization of the functions and proteomes associated with membrane rafts in chicken sperm

Abstract

Cellular membranes are heterogeneous, and this has a great impact on cellular function. Despite the central role of membrane functions in multiple cellular processes in sperm, their molecular mechanisms are poorly understood. Membrane rafts are specific membrane domains enriched in cholesterol, ganglioside GM1, and functional proteins, and they are involved in the regulation of a variety of cellular functions. Studies of the functional characterization of membrane rafts in mammalian sperm have demonstrated roles in sperm-egg binding and the acrosomal reaction. Recently, our biochemical and cell biological studies showed that membrane rafts are present and might play functional roles in chicken sperm. In this study, we isolated membrane rafts from chicken sperm as a detergent-resistant membranes (DRM) floating on a density gradient in the presence of 1% Triton X-100, and characterized the function and proteomes associated with these domains. Biochemical comparison of the DRM between fresh and cryopreserved sperm demonstrated that cryopreservation induces cholesterol loss specifically from membrane rafts, indicating the functional connection with reduced post-thaw fertility in chicken sperm. Furthermore, using an avidin-biotin system, we found that sperm DRM is highly enriched in a 60 KDa single protein able to bind to the inner perivitelline layer. To identify possible roles of membrane rafts, quantitative proteomics, combined with a stable isotope dimethyl labeling approach, identified 82 proteins exclusively or relatively more associated with membrane rafts. Our results demonstrate the functional distinctions between membrane domains and provide compelling evidence that membrane rafts are involved in various cellular pathways inherent to chicken sperm.

Src family kinases-mediated negative regulation of sperm acrosome reaction in chickens (Gallus gallus domesticus)

“…Studies in other cells demonstrated the functional and spatial associations of SFKs, with MRs playing significant roles in an assortment of cellular processes [ 57 ]. In addition to previous findings that MRs are present in mammalian sperm and regulate multi-stage fertilization [ 58 , 59 ], our results have revealed that chicken sperm possess membrane domains that play an important role in AR induction and binding to IPVL [ 11 , 16 , 60 ]. This study found a relative abundance of c-Src and p-SFK-Y416 in TX-100 insoluble membrane fractions that compositionally mimic MRs, which was different from previous results in which bull sperm have more enriched p-SFK-Y416 in non-rafts than MRs [ 61 ].…”

Testis-Specific Isoform of Na+-K+ ATPase and Regulation of Bull Fertility

“…The presence of raft and non-raft fraction in sperm plasma membrane has been widely reported in sperm from mice [ 88 ], pigs [ 89 ], bulls [ 20 ], chickens [ 90 ], and humans [ 91 ]. Although cholesterol helps in lipid raft stabilization, regulated low-level cholesterol efflux from the sperm plasma membrane during capacitation does not affect the raft composition [ 92 , 93 ].…”

“…Moreover, lipid rafts enrich a variety of ion transporters/channels. For instance, plasma membrane Ca 2+ ATPase is enriched in sperm lipid raft fractions from chickens [ 90 ], bulls [ 109 ], mice [ 98 ], and humans [ 99 ], which significantly contributes to the induction of an acrosome reaction and hyperactivated motility [ 110 ].…”

“…Another vital ion transporter in the sperm plasma membrane is NKA, which mainly resides in lipid rafts, facilitating cell signaling due to its proximity to other signaling molecules within these microdomains/rafts [ 30 ]. Ushiyama et al [ 90 ] reported that lipid rafts in the chicken sperm membrane were enriched in NKA isoforms (α1, α3, β1). Various NKA isoforms involved in capacitation-associated signaling have been demonstrated in both raft and non-raft fractions of the plasma membrane of bull sperm.…”

“…Moreover, it is argued that lipid rafts allow activated receptors enhanced access to specific downstream signaling proteins involved in signal transduction, and interactions with unrelated proteins [87]. The presence of raft and non-raft fraction in sperm plasma membrane has been widely reported in sperm from mice [88], pigs [89], bulls [20], chickens [90], and humans [91]. Although cholesterol helps in lipid raft stabilization, regulated low-level cholesterol efflux from the sperm plasma membrane during capacitation does not affect the raft composition [92,93].…”