Importance of Ca2+, K+ and Glucose in the Medium for Sperm Penetration through the Human Zona Pellucida.

Abstract

When salt-stored human oocytes were inseminated in the regular mBWW medium and examined 6 hr later, the zonae of almost all of the oocytes were penetrated by spermatozoa. In contrast, none of zonae were penetrated when the oocytes were inseminated in either Ca2+-free, K+-free or glucose-free medium. The failure of zona penetration by spermatozoa in Ca2+-free and K+-free media is likely to be due to the inhibition of the acrosome reaction. Spermatozoa were unable to exhibit hyperactivated motility in glucose-free medium, suggesting that the failure of zona penetration in this medium is due, at least in part, to the inhibition of hyperactivated motility of spermatozoa.calcium ; potassium ; glucose ; sperm ; zona pellucidaUnder normal conditions mammalian spermatozoa must pass through the zona pellucida before effecting fertilization.Proper interaction of sperm molecules with the complementary zona molecules, strong flagellar motion of the spermatozoa and ionic balance of the medium are all essential for successful penetration of spermatozoa through the zona pellucida (O'Rand et al. 1986;Yanagimachi 1988;Saling 1989;Wassarman 1990). We report here that human spermatozoa are unable to attach to and penetrate through the zona unless the medium contains millimolar concentrations of Ca2+, K+ and glucose. MATERIALS AND METHODS MediaThe standard medium used in this study was a modified Krebs-Ringer's solution, mBWW (Overstreet et al. 1980) containing 35 mg/ml human serum albumin (HSA ; Sigma Chemical Co., St. Louis, MO, USA). This medium had an osmolality of 308 mOsmol and pH value of 7.5±0.1 when equilibrated with 5% CO2 in air. Ca2+-free, Mg2+-free, glucosefree, lactate-free and pyruvate-free media were prepared by replacing CaC12i MgSO4, glucose, Na-lactate and Na-pyruvate, respectively, with appropriate amounts of NaC1 to keep the osmolality of the media constant. K+-free medium was prepared by replacing KCl and KH2 PO4 with NaCI and NaH2 PO4 i respectively. The pH of these media was adjusted, if necessary, to 7.5 by adding a small quantity of 1 M HC1 or NaOH. All media were

Fibrous sheath of mammalian spermatozoa

“…Hyperactivation is the change in motility from low amplitude, progressive motility seen in freshly ejaculated or diluted epididymal sperm to a high-amplitude whiplash flagellar beat (Yanagimachi, 1994). It is thought to facilitate sperm transport in the oviduct and penetration of the zona pellucida (reviewed in Suarez, 1996) and glucose is required for hyperactivation and zona pellucida penetration by mouse (Fraser and Quinn, 1981;Cooper, 1984;Urner and Sakkas, 1996) and human sperm (Hoshi et al, 1991). Other energy substrates, including lactate, pyruvate, and glucose analogs that cannot be metabolized did not support hyperactivation in these studies, indicating a specific requirement for glycolysis.…”

“…This cytoskeletal structure is ideally positioned to serve as a scaffold for glycolytic enzymes, providing a mechanism for localized ATP production along the axoneme. Although ATP is also produced in the midpiece mitochondria, production of ATP via glycolysis is required for hyperactivated motility (Hoshi et al, 1991;Urner and Sakkas, 1996).…”

Cleavage of Disulfide Bonds in Mouse Spermatogenic Cell-Specific Type 1 Hexokinase Isozyme Is Associated with Increased Hexokinase Activity and Initiation of Sperm Motility1

“…Although oxidative phosphorylation is the main source and glycolysis is a minor source of ATP in most cell types, fertilization was not prevented by pharmacological inhibition of oxidative phosphorylation [22,24] or disruption of the testis-specific cytochrome C (Cyct) gene [25]. However, in vitro fertilization failed if the medium lacked glucose [24,[26][27][28][29], suggesting that glycolysis produces most of the ATP needed for sperm function. This was shown convincingly by using gene targeting to disrupt the sperm-specific Gapdhs gene.…”

Moving to the beat: a review of mammalian sperm motility regulation

“…It has also been shown that mammalian sperm produce lactate from glucose under aerobic conditions (Storey and Kayne 1975). Adenosine triphosphate production through glycolysis is required for hyperactivated sperm motility (Hoshi et al 1991;Urner and Sakkas 1996) and inhibition of oxidative phosphorylation does not block fertilisation (Fraser and Quinn 1981).…”