The site of gamete interaction of electrophysiologically recorded Lytechinus variegatus eggs, fixed with osmium tetroxide (O5O4) and/or glutaraldehyde (GTA) at varying intervals after the onset of the increase in membrane conductance induced by an attached sperm, has been examined by high-voltage and conventional transmission electron microscopy. Although GTA and a GTA-O5O4 mixture iduced different electrical responses, specimens prepared with the two fixatives were ultrastructurally similar. In specimens observed within 5 s of the change in conductance, the acrosomal process projected through the vitelline layer and abutted the egg plasma membrane. A conspicuous layer of bindin surrounded the acrosomal process and connected the sperm to the egg's vitelline layer. In a fortuitous specimen fixed within 4 s following the change in conductance, the area of contact between the gamete plasma membranes possessed a trilaminar structure that separated the egg's and sperm's cytoplasms. The morphology of this area of contact was consistent with previously proposed intermediates of membrane fusion. Five to six seconds after the change in conductance, the sperm was connected to the egg via a narrow cytoplasmic bridge that consisted of the former acrosomal process and a projection of the egg cortex. The region of the bridge midway between the fused gametes was encircled by dense material that marked the site of sperm-egg fusion. Gamete interactions in which the activation potential was recorded (unclamped egg) were comparable in time and ultrastructure to events taking place in voltage-clamped eggs except for one major difference. Intact cortical granules (one to three) were observed beneath the tip of the incorporating sperm in unclamped eggs fixed following the onset of the activation potential, whereas all cortical granules dehisced in clamped eggs.

Isolated seminiferous tubules of rat testis contain considerable urokinase-inhibiting activity. An immunohistological analysis revealed the presence of plasminogen activator inhibitor type 1 (PAI-1) in the basement membrane as well as in the interior of the tubules. Distribution and intensity of the intratubular immunoreactivity depends on the tubules. Distribution and intensity of the intratubular immunoreactivity depends on the stage of the seminiferous cycle. A relatively weak signal is present around elongated nuclei of spermatids at the beginning of chromatin condensation. The signal intensity increases in the course of differentiation until a maximum is reached at stages VII-VIII. In these stages PAI-1 immunoreactivity is localised around the nuclei of the late spermatids as well as along their tails. Spermatozoa in the ductus epididymis also strongly react with the PAI-1-specific antiserum, suggesting that the inhibitor remains associated with the germ cells after spermiation and during maturation in the epididymis. In intact mature spermatozoa isolated from epididymis cauda by ‘swimming-up’ in non-capacitation medium, PAI-1 antigen is localised on the plasma membrane surrounding the head. In addition, in fixed and permeabilised cells the immunoreactivity is detectable in the acrosome and in the tail. Possible functions of PAI-1 in spermatogenesis, sperm motility and sperm-egg interaction are discussed.

Sperm plasma membrane (PM) proteins that demonstrate affinity for egg PM preparations have the potential to be biologically important during sperm-egg binding and/or fusion. In this study four such proteins have been identified. To provide quantitative evidence for possible biological function, the large natural variation among different porcine sperm populations with regard to their ability to interact with the egg was compared with the relative binding of egg PM material to individual proteins. An aliquot from each of 24 porcine ejaculates was evaluated by the zona-free hamster ova bioassay and the remainder processed to yield sperm PM vesicles. Aliquots of sperm PM were solubilised, separated by SDS-PAGE, western blotted and probed with partially purified, biotinylated egg PM protein. Bound egg PM proteins were visualised on western blots by an avidin/biotin/horseradish peroxidase system and analysed by scanning laser densitometry. Four sperm PM proteins (62, 39, 27 and 7 kDa estimated molecular mass) were the predominant binders of egg PM. The amount of egg PM bound to the 62 kDa protein was significantly correlated with the ability of sperm from the 24 ejaculates to penetrate zona-free hamster ova (percentage of ova penetrated, p = 0.01, R = 0.65; number of penetrated sperm per ovum, p = 0.02, R = 0.63)

In vitro fertilisation (IVF) was used to assess the ability of solubilised sperm plasma membrane (PM) proteins to inhibit the interaction of intact gametes. This is a first step before evaluating the ability of individual isolated proteins to competitively inhibit sperm-oocyte interaction as part of the process of studying the molecular events of fertilisation. Porcine oocytes were aspirated from ovaries, matured for 48 h in Medium 199, and the zona pellucida (ZP) was removed by exposure to acid Tyrode's solution. ZP-free matured oocytes were exposed to 200–800 μg/ml sperm PM protein for 1 h prior to insemination and during gamete co-incubation. Twenty-four hours after insemination with 5 × 105 capacitated sperm/ml, the oocytes were fixed, stained and examined. Sperm PM protein clearly inhibited IVF in a concentration-dependent manner (r = −0.87). The inhibition index (I50%), representing the sperm PM protein concentration necessary to inhibit IVF to 50% of the control value, was 310 µg/ml. These results demonstrate that solubilised sperm PM protein inhibits the interaction of intact gametes as one might expect for receptor-ligand interactions. Furthermore, the complement of sperm PM proteins appeared maximally effective at a calculated concentration of 690 µm/ml, providing a foundation for further studies with individual proteins.