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Visualization of G_M1 with Cholera Toxin B in Live Epididymal Versus Ejaculated Bull, Mouse, and Human Spermatozoa¹

The James A. Baker Institute for Animal Health,³ College of Veterinary Medicine, Cornell University, Ithaca, New York 14853 Weill Medical College,⁴ Cornell University, New York, New York 10021 Population Council,⁵ The Rockefeller University, New York, New York 10021

ABSTRACT

The organization of membrane subdomains in mammalian sperm has recently generated controversy, with several reports describing widely differing localization patterns for the ganglioside G_M1. Using the pentameric B subunit of cholera toxin (CTB), we found G_M1 to be restricted to the plasma membrane overlying the acrosome in the heads of live murine sperm. Interestingly, CTB had minimal binding to live bovine and human sperm. To investigate whether this difference in G_M1 localization was because of species differences or differences between collection from the epididymis (mouse) or an ejaculate (bull, human), we examined epididymal bovine and human sperm. We found that G_M1 localized to the plasma membrane overlying the acrosome in sperm from these species. To determine whether some component of seminal plasma was interfering with the ability of CTB to access G_M1, we incubated epididymal mouse sperm with fluid from murine seminal vesicles and epididymal bull sperm with bovine seminal plasma. This treatment largely abolished the ability of the CTB to bind to G_M1, producing a fluorescence pattern similar to that reported for the human. The most abundant seminal plasma protein, PDC-109, was not responsible for this loss. As demonstration that the seminal plasma was not removing G_M1, sperm exposed to seminal plasma were fixed before CTB addition, and again displayed fluorescence over the acrosome. These observations reconcile inconsistencies reported for the localization of G_M1 in sperm of different species, and provide evidence for the segregation of G_M1 to a stable subdomain in the plasma membrane overlying the acrosome.

epididymis, gamete biology, seminal vesicles, sperm

¹ Supported by National Institutes of Health grants R01-HD-045664, K01-RR00188 (A.J.T.), and R01-HD-038807 (G.R.H.), a Genex/CRI grant (A.J.T), and the Cornell College of Veterinary Medicine DVM/Ph.D. Dual Degree Program (D.E.B.).

² Correspondence. FAX: 607 256 5608; ajt32{at}cornell.edu

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