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This study characterizes a common antigen recognized by two monoclonal antibodies (mAbs) that immunoreact with the principal piece of the human sperm flagellum. By means of immunofluorescence microscopy, mAbs S69 (IgM) and S70 (IgG1) (WHO Task-force nomenclature) were observed to bind to the principal piece of methanol- or detergent-permeabilized human spermatozoa, but did not react with live swimming spermatozoa as assessed by immunofluorescence microscopy. Faint immunofluorescence was also seen on the connecting piece in approximately 40% of the spermatozoa. Immunoreactivity in both regions was resistant to sequential extraction with Triton X-100, sodium thiocyanate, and urea. Pre-embedding electron microscopic immunogold labeling of ejaculated spermatozoa with mAb S69 showed gold particles located on the fibrous sheath. Immunoreactive peptides of 68, 53, and 45 kDa were recognized by both S69 and S70 mAbs on immunoblots of nonreduced human sperm extracts, while a 68-kDa band and a strongly immunoreactive triplet from 54 to 51 kDa were recognized in reduced sperm extracts. Human fibrous sheaths were isolated by differential solubilization and centrifugation and characterized by transmission electron microscopy. The 68-kDa and 54-51-kDa bands were enriched and found to be major polypeptides in the isolated fibrous sheath fraction. These results suggest that the S69/S70 antigen, which we term SP (sperm protein) (68 kDa, 54-51 kDa), is a component of the human fibrous sheath.

Sperm antigens recognized by monoclonal antibodies (mAbs), S19, S69, S71, S72 and S77 submitted to the World Health Organization (WHO)-Sponsored Sperm Monoclonal Antibody Workshops were immunolocalized by light (LM) and transmission electron microscopy (TEM). S19 was surface reactive while mAbs S69, S71, S72 and S77 recognized internal antigens. Indirect immunofluorescence staining of permeabilized sperm with the mAbs revealed that S69 recognized an internal tail antigen, while S71, S72 and S77 recognized acrosomal proteins. Preservation of immunoreactivity after fixation in various combinations of glutaraldehyde, paraformaldehyde and tannic acid was evaluated for mAbs S69 to S77 using immunofluorescence microscopy. The epitopes recognized by these mAbs were adversely affected by these fixatives; therefore, pre-embedding immunogold staining was employed, prior to fixation, osmication, dehydration and embedding. Using this approach, the antigen recognized by mAb S19 was found associated with the plasmalemma of the head and tail of intact sperm. Monoclonal antibody S69 localized to the fibrous sheath. The mAbs S71, S72 and S77, which required sperm permeabilization to expose their acrosomal locus by LM, did not immunoreact with the plasmalemma at the TEM level. Ultrastructural examination of acrosome-reacted sperm revealed the localization of S71 and S77 on the inner and outer acrosomal membranes and with acrosomal matrix. The S72 antigen was associated with the inner and outer acrosomal membranes.

The human sperm protein SP-10 was previously defined as a "primary vaccine candidate" by a World Health Organization Taskforce on Contraceptive Vaccines. By one- and two-dimensional immunoblots, we show that SP-10, extracted from ejaculated human sperm, demonstrated a polymorphism of immunogenic peptides from 18 to 34 kDa, a pattern that was conserved from individual to individual and was not altered by reducing agents. The majority of the antigenic peptides possessed isoelectric points of approximately 4.9. Immunocytochemistry on testis sections indicated that SP-10 was localized to round spermatids and spermatozoa within the adluminal compartment of the seminiferous epithelium. Immunofluorescence showed that SP-10 was not associated with the surface of acrosome-intact, ejaculated sperm. Light and electron microscopic immunocytochemistry localized SP-10 throughout the acrosome, and electron microscopic evidence demonstrated a bilaminar array in association with the inner aspect of the outer acrosomal membrane and the outer aspect of the inner acrosomal membrane. After induction of the acrosome reaction with the ionophore A23187, SP-10 remained displayed on the sperm head in association with the inner acrosomal membrane and equatorial segment. The results indicate that the MHS-10 monoclonal antibody may be used as a marker of acrosome development in the human and as a probe to evaluate acrosome status. The results also support the hypothesis that inhibition of sperm-egg interaction by anti-SP-10 monoclonal antibody may occur as a result of antigen exposure following the acrosome reaction.

The monoclonal antibody (mAb) MHS-10 (IgG1) is a mouse antihuman sperm antibody which recognizes a polymorphic sperm protein, (SP-10), which has previously been localized within the acrosomal matrix and the acrosomal membranes. The SP-10 antigen has been shown to be sperm-specific and is not found in somatic tissues. It is stage specific, having been immunohistologically localized to Golgi phase spermatids and all subsequent phases of spermiogenesis. In the present study, acetone-dried smears from washed human semen containing significant numbers of round cells were probed with mAb MHS-10. Monoclonal antibody-labeled cells were visualized by a standard streptavidin-biotin immunoperoxidase method using a light microscope. The MHS-10 mAb immunoreacted with mature sperm and with a subset of round cells diagnosed as developing spermatids, which had been sloughed off from the testis at varying stages of acrosome formation. To rule out possible cross-reactivity of the mAb with leukocytes in semen, a leukocyte surface marker (anti-HLe-1) was used in conjunction with MHS-10. Round cell populations staining with MHS-10 did not stain with anti-HLe-1. The mAb MHS-10 is thus a promising probe for the identification and quantitation of immature germ cells in human semen.

Kinetic measurements indicate that the energy-independent transhydrogenation of 3-acetylpyridine-NAD+ by NADPH in membranes of Escherichia coli follows a rapid equilibrium random bireactant mechanism. Each substrate, although reacting preferentially with its own binding site, is able to interact with the binding site of the other substrate to cause inhibition of enzyme activity. 5'-AMP (and ADP) and 2'-AMP interact with the NAD+- and NADP+-binding sites, respectively. Phenylglyoxal and 2,3-butanedione in borate buffer inhibit transhydrogenase activity presumably by reacting with arginyl residues. Protection against inhibition by 2,3-butanedione is afforded by NADP+, NAD+, and high concentrations of NADPH and NADH. Low concentrations of NADPH and NADH increase the rate of inhibition by 2,3-butanedione. Similar effects are observed for the inactivation of the transhydrogenase by tryptic digestion in the presence of these coenzymes. It is concluded that there are at least two conformations of the active site of the transhydrogenase which differ in the extent to which arginyl residues are accessible to exogenous agents such as trypsin and 2,3-butanedione. One conformation is induced by low concentrations of NADH and NADPH. Under these conditions the coenzymes could be reacting at the active site or at an allosteric site. The stimulation of transhydrogenase activity by low concentrations of the NADH is consistent with the latter possibility.