Changes to the spermatozoa glycocalyx and its role in fertilization in Sauger (Sander canadensis)

Abstract

The spermatozoa glycocalyx is a dynamic coating of extracellular glycoproteins known to facilitate the acrosome reaction and fertilization in mammals. Most fish sperm, however, contain no acrosome and are subjected to different stimuli (e.g., osmotic shock in the external environment) prior to fertilization. At present, our understanding of the composition and spatial distribution of sugar moieties and the functional role of the glycocalyx in fish sperm remains incomplete. Moreover, the influence of assisted reproduction techniques (e.g., testicular harvest and cryopreservation) commonly used in aquaculture settings on this structure in fish sperm is unknown. Herein, we describe and compare the composition and characteristics of the glycocalyx among sperm types (stripped, testicular, and cryopreserved) and between activation statuses (inactive vs. activated) using sauger (Sander canadensis) as a model. We also investigated the importance of certain moieties (e.g., N-acetyl-glucosamine [GlcNAc]) to fertilization. Staining distributions, fluorescent intensity, and proportion of cells exhibiting high fluorescence were measured for each treatment using fluorescent microscopy and flow cytometry, respectively. Three lectins, specific to certain glycocalyx sugar moieties (wheat germ agglutinin, WGA [GlcNAc]; concanavalin A, ConA [α-mannose]; and peanut agglutinin, PNA [β-galactose]) were used to monitor these variables in fish sperm. The sauger glycocalyx contained GlcNAc and α-mannose but lacked β-galactose moieties. Testicular sperm exhibited fewer cells (20–40% fewer) with high GlcNAc and α-mannose content than other sperm types. Additionally, the proportion of highly fluorescent cells in testicular sperm were positively correlated with motility (r = 0.80–0.95), suggesting the glycocalyx is affected by post-testicular maturation. Motility activation via hypo-osmotic shock caused a redistribution of GlcNAc to the apical region of the head of 40–50% of stripped and testicular sperm, respectively. Cryopreserved sperm showed significantly reduced apical staining following activation (< 5%) and a 2 to 3-fold increase in α-mannose availability. Further, fertilization was reduced by ~80% compared to a fresh control (i.e., untreated stripped sperm) in both stripped sperm pre-treated block GlcNAc during insemination and cryopreserved sperm. These results indicate that the glycocalyx of sauger sperm is dynamic, as it is modified during post-testicular maturation, motility activation, and that it plays a pivotal role in fertilization. Cryopreservation largely negated these changes, which may partially explain the reduced fertility observed in frozen sperm. Ultimately, our analyses show that the use of lectins (e.g., GA and ConA) as biomarkers for the glycocalyx can help to understand the quality and fertilization potential of fish sperm.