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Lead remains in high levels in the environment and is known to reduce fertility in animal models, but a direct link between lead exposures and human infertility has not yet been established.

Normozoospermic men who do not fertilize human eggs in vitro constitute a clearly identifiable class of occult male factor infertility. We have studied the relationships between in-vitro fertilization (IVF) outcomes and the appearance of sperm surface mannose lectin/zona pellucida ligand activity measured by the binding of fluorescein isothiocyanate (FITC)-labelled mannosylated albumin. Of 338 semen specimens with normal profiles, 10 (2.96%) failed to fertilize in vitro (IVF−). Motile spermatozoa from six of these were analysed retrospectively for zona ligand binding activity on the sperm head in relation to acrosome status and the free cholesterol content of their plasma membrane. IVF+ males and fertile donors served as controls. The six IVF− occult male factor cases were distinguished from controls by their failure to show time-dependent increases in the percentage of spermatozoa exhibiting head-directed mannose ligand activity, by the slow loss or apparent increase in membrane cholesterol and by the absence of spontaneous and/or mannose-induced acrosome exocytosis.

D-Mannose binding lectins appear on the human sperm head following in-vitro incubation under capacitating conditions. Surface expression of lectin is dependent on reduction of the sterol content of sperm membranes. Mannose-specific lectin distribution over the head differs in acrosome-intact and acrosome-reacted spermatozoa. Sugar competition experiments show that mannose is the only saccharide competitive with D-mannosylated albumin binding and that free mannose induces an acrosome reaction in capacitated spermatozoa. A total of 183 men with normozoospermic semen parameters were screened for the ability of their spermatozoa to bind fluorescein isothiocyanate (FITC)-labelled mannosylated albumins, before and after incubation in vitro. The spermatozoa from 176 men ‘responded’ to incubation by exhibiting time-dependent increases in head-directed mannosylated albumin binding, accompanied by increases in the percentage of spermatozoa showing spontaneous acrosome loss. Motile spermatozoa from the remaining seven men failed to express mannose—lectin binding activity after 18 h of incubation and only low percentages of their spermatozoa showed spontaneous acrosome loss. These seven men were classified as ‘non-responders’. The relative amounts of non-esterified cholesterol in the sperm membranes of the responder and non-responder males were analysed by gas—liquid chromatography. Responder spermatozoa showed decreases in free cholesterol content whereas non-responder spermatozoa exhibited either no decrease or increases in relative free cholesterol per cell. Fresh swim-up spermatozoa contain sub-plasma membrane stores of mannose lectins which are revealed by FITC-mannosylated albumin labelling before and after removal of the plasma membrane by vortexing. In contrast, the mannose—lectin binding activity of capacitated spermatozoa is entirely limited to the sperm surface. Western blots of proteins isolated from sperm plasma membranes after capacitation revealed two molecular species reactive with polyclonal antibodies against human macrophage mannose receptors. A model is proposed for the molecular mechanism whereby mannose lectins are transposed from sub-plasma membrane sites to an integral membrane position as a consequence of the loss of cholesterol from the sperm membrane.

Polyvalent mannose ligands in the presence of free mannose act as zona pellucida agonists which rapidly induce acrosome exocytosis in competent motile human sperm from fertile donors following in-vitro capacitation. Quantification of the binding patterns of fluorescein isothiocyanate labelled mannosylated albumins and of specific antisera which recognize mannose receptors and other related integral sperm membrane proteins as well as the incidence of induced acrosome exocytosis after capacitation has allowed us to identify three categories of male infertility. Category 1 males have normozoospermic semen parameters, their spermatozoa have elevated sperm cholesterol values and fail to fertilize oocytes in vitro after standard short-term incubations. These spermatozoa do not bind mannose ligands and do not show spontaneous or induced acrosome reactions, but treatments to remove cholesterol from the spermatozoa (e.g. prolonged incubation in the presence of sterol acceptors) confer the ability to fertilize. Cholesterol loading and unloading experiments have demonstrated the reversible character of sperm membrane properties in category 1 male infertility. Category 2 males have normal-appearing spermatozoa in semen which express mannose ligand receptors on incubation, but fail to undergo acro some reactions in response to mannose treatment. Interestingly, all category 2 males identified in this study have clinical varicocele. Category 3 males have semen which may be normozoospermic or teratozoospermic with, in some cases, high percentages of tapering spermatozoa in the absence of clinical varicocele. Spermatozoa from category 3 men are deficient in a superfamily of integral membrane proteins whose cytoplasmic tails have myosin motors as identified by amino acid sequence analysis and anti-myosin antibody reactivity. Their spermatozoa do not express mannose ligand receptors or undergo induced acrosome reactions. Fertilization with category 2 and 3 semen is only achieved by micromanipulation procedures. These findings illustrate the practical application of basic research for infertility classification.