Abstract
Sperm capacitation in vitro is highly correlated with an increase in protein tyrosine phosphorylation that is regulated by cAMP through a unique mode of signal transduction cross-talk. The activation of this signaling pathway, as well as capacitation, requires bovine serum albumin (BSA) in the incubation medium. BSA is hypothesized to modulate capacitation through its ability to remove cholesterol from the sperm plasma membrane. Here we demonstrate that the cholesterol-binding heptasaccharides, methyl-beta-cyclodextrin and OH-propyl-beta-cyclodextrin, promote the release of cholesterol from the mouse sperm plasma membrane in media devoid of BSA. Both of these beta-cyclodextrins were also demonstrated to increase protein tyrosine phosphorylation in the absence of BSA in both mouse and bull sperm, and the patterns of phosphorylation were similar to those induced by media containing BSA. The potency of the different beta-cyclodextrins to increase protein tyrosine phosphorylation in sperm was correlated with their cholesterol binding efficiencies, and preincubation of the beta-cyclodextrins with cholesterol-SO4- to saturate their cholesterol-binding sites blocked the ability of these compounds to stimulate protein tyrosine phosphorylation. The beta-cyclodextrin effect on protein tyrosine phosphorylation was both NaHCO3 and protein kinase A-dependent. The beta-cyclodextrins were also able to capacitate mouse sperm in the absence of BSA, as measured by the ability of the zona pellucida to induce the acrosome reaction and by successful fertilization in vitro. In summary, beta-cyclodextrins can completely replace BSA in media to support signal transduction leading to capacitation. These data further support the coupling of cholesterol efflux to the activation of membrane and transmembrane signaling events leading to the activation of a unique signaling pathway involving the cross-talk between cAMP and tyrosine kinase second messenger systems, thus defining a new mode of cellular signal transduction initiated by cholesterol release.
Highlights
Sperm capacitation in vitro is highly correlated with an increase in protein tyrosine phosphorylation that is regulated by cAMP through a unique mode of signal transduction cross-talk
Release of Cholesterol from the Sperm Plasma Membrane in Media containing bovine serum albumin (BSA), 2-OH-propyl--cyclodextrin, or Methyl-cyclodextrin—Previously, we demonstrated in mouse sperm that both protein tyrosine phosphorylation and capacitation required the presence of BSA in the incubation medium [15]
This hypothesis would predict that other cholesterol binding compounds that remove cholesterol from the sperm plasma membrane could substitute for BSA in media to regulate signal transduction leading to capacitation
Summary
Materials—2-OH-propyl--cyclodextrin, methyl--cyclodextrin, and BSA (Fraction V, Sigma) were purchased from Sigma. Steroid Measurements—Mouse sperm (5 ϫ 106) were incubated in 500 l of capacitation medium in either the absence or presence of 3 mg/ml BSA or with the appropriate -cyclodextrins for 1.5 h After this period, each aliquot was centrifuged for 10 min at 10,000 ϫ g and cholesterol, desmosterol, and cholesterol-SO4Ϫ were measured in the sperm pellet and in the resultant medium supernatant as described previously [26]. The resultant supernatants following the initial centrifugation were extracted with 6 volumes of chloroform/methanol (2:1, v/v), vortexed for 10 s, centrifuged at 800 ϫ g for 10 s, and the lower organic phase aspirated and evaporated to dryness Both the sperm pellet and medium supernatant extracts were dissolved in 20 l of chloroform/methanol (1:1, v/v), and 4-l aliquots applied to silver nitrate-impregnated Whatman HP-K silica gel microplates (Whatman Inc., Clifton, NJ) Phase-contrast optics were used to evaluate fertilization by looking for the presence of the second polar body and the formation of both the male and female pronuclei
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