Changes in binding of lectins to epididymal, ejaculated, and capacitated spermatozoa of the rhesus monkey

Abstract

Background The present study was undertaken to evaluate the changes in rhesus monkey sperm surface glycoconjugates during maturation, ejaculation, and capacitation in order to provide background information that would help in evaluating adverse effects, if any, caused by the use of contraceptive agents. Methods Adult sexually mature rhesus monkeys were castrated under ketamine anaesthesia. Percoll purified sperm from different epididymal segments and motile‐ejaculated spermatozoa prior to and following in vitro capacitation were exposed to FITC‐labeled lectins. Live or sperm prefixed with paraformaldehyde/alcohol were used. Quantitative analysis of changes in lectin binding was done by immunoassay. Results The majority of live spermatozoa did not show binding of Con A and PNA, whereas uniform labeling of WGA to sperm from corpus epididymidis onward was seen. Unfixed spermatozoa showed marked variation in the pattern of lectin binding. The majority of spermatozoa fixed with paraformaldehyde or alcohol showed maximum lectin labeling over the acrosome, but the postacrosomal area invariably did not bind the lectins. Major changes in the localization of Con A, PNA, or WGA to prefixed spermatozoa were not seen during epididymal transit and in the ejaculate. In capacitated acrosome‐reacted spermatozoa, Con A, PNA, and WGA were localized mainly in the equatorial segment. Binding of Con A and PNA was abolished by using lectins preincubated with appropriate inhibitor saccharides. Sperm, exposed to FITC‐WGA preincubated with inhibitor sugar, did not show complete inhibition. Quantitative analysis of lectin binding by immunoassay showed increase in binding of lectins during epididymal transit with maximum binding in sperm from the corpus epididymidis. Conclusions The variations in lectin labeling of live spermatozoa could be due to redistribution of sperm surface sugars or membrane damage. The changes in lectin labeling during maturation and capacitation may be associated with their role in ovum recognition and fusion. © 1996 Wiley‐Liss, Inc.

Live Rhesus Offspring by Artificial Insemination Using Fresh Sperm and Cryopreserved Sperm1

“…The high affinity of FITC-PNA for the acrosomal region makes this lectin a useful probe with which to determine acrosome integrity [11,21,22]. In the present study, FITC-PNA was localized on the acrosomal cap of sperm only, and this finding is similar to that reported for ejaculated rhesus sperm by Navaneetham et al [11].…”

“…Sperm were attached to poly-L-lysine-coated microscopy coverslips by a 5-min incubation in a 1-ml drop of warm PBS and then fixed for 1 h in 2% formaldehyde in PBS. Following a rinse in PBS, sperm were incubated with 100 l of PBS containing 1 g of fluorescein isothiocyanate-conjugated peanut lectin (FITC-PNA; Sigma) in the dark for 1 h at room temperature [11]. Next, 2 l of PBS containing 10 g of 4Ј,6Ј-diamindino-2-phenylindole (DAPI; Molecular Probes, Eugene, OR) were added to the FITC-PNA solution 5 min before the end of incubation.…”

Partial characterization, sperm association and significance of N- and O-linked glycoproteins in epididymal fluid of rhesus monkeys (Macaca mulatta)

“…Lectins have been widely used as probes in the examination of glycocomponents of the cell surface and secretions in a variety of species (Young et al 1986, Arenas et al 1996, Navneetham et al 1996, Calvo et al 2000. The use of lectins to probe Western blots of SDS-PAGE-fractionated plasma membranes of spermatozoa from various segments of the epididymis in mouse, rat, rhesus monkey and human spermatozoa (Rankin et al 1989, Srivastava & Olson 1991, Liu et al 2000, Srivastav 2000) examines a wide diversity of carbohydrate moieties and permits direct analysis of glycocomponents on Western blots of fractionated proteins, within specific epididymal segments and, furthermore, provides evidence for different mechanisms that may generate these changes.…”

“…Although the characterization, significance and role of epididymal glycoproteins have been studied in many primate species including human (Focarelli et al 1998, Kirchhoff et al 1998, Martin Ruiz et al 1998, Liu et al 2000, Yeung et al 2000b, these are poorly understood in the epididymis of the rhesus monkey (Navneetham et al 1996), an animal model commonly used for preclinical testing of drugs. Previous work from this laboratory has demonstrated maturation-dependent changes in the glycoprotein profile of purified plasma membrane of spermatozoa from rhesus monkey epididymis, and a few of these glycoproteins co-migrated with proteins present in the caudal epididymal fluid.…”

Evidence for the involvement of PECAM-1 in a receptor mediated signal-transduction pathway regulating capacitation-associated tyrosine phosphorylation in human spermatozoa

“…Given the complexity of the sperm glycocalyx, this cell type has proved particularly amenable to studies involving lectins. As such, there is an extensive literature documenting the use of these probes to study changes in sperm surface architecture that accompany key biological events such as epididymal maturation and the capacitation of these cells within the female reproductive tract (Bearer and Friend, 1990;Gall et al, 1975;Koehler, 1978;Koehler, 1981;Lee and Damjanov, 1985;Mahmoud and Parrish, 1996;Navaneetham et al, 1996;Nicolson et al, 1977;Nicolson and Yanagimachi, 1974;Olson and Danzo, 1981). More recently, it has also been demonstrated Mammalian spermatozoa must become 'capacitated' in the female reproductive tract before they gain the ability to fertilize the oocyte.…”