Structure of sperm activating protein.

Serum is used as an additive in the preparation of human spermatozoa for fertilization in vitro, as it is superior to other body fluids in supporting sperm motility. We recently purified the major sperm activating macromolecule present in serum and showed it to be a complex of immunoglobulin and apolipoprotein A-I. This complex, which we named sperm activating protein (SPAP), has now been further characterized using partial proteolysis in combination with different immunological methods. SPAP was shown to interact only with antibodies against immunoglobulin G and more specifically with those against IgG4. The bacterial expression products C23 and ZZ-T (which bind to specific sites on the IgG molecule) bound in similar ways to SPAP as to IgG4 and did not hinder proteolytic cleavage of SPAP, indicating that apolipoprotein A-I is not bound closely to the binding sites of these proteins. Purified F(ab')2 fragment from SPAP was also shown to contain apolipoprotein A-I, and had a higher MW than the corresponding fragment from IgG4. Taken together, the most plausible (and in our view only possible) structure of SPAP shows an apolipoprotein A-I molecule bound in the pocket formed between the Fab arms of an IgG4 molecule. Anti-SPAP antibodies visualized by secondary fluorescein isothiocyanate (FITC)-labelled antibodies were bound to the postacrosomal part of the spermatozoa, indicating that SPAP is specifically bound to this area, and directly interacts with the spermatozoa. Based on these and earlier experiments it is speculated that SPAP acts in the lower part of the female genital tract. The benefit of SPAP should be at its greatest in these regions, and it is also possible that SPAP exerts a selection mechanism, as those spermatozoa affected by SPAP acquire increased motility, which might be important in order to reach the upper part of the female genital tract. Further exploration of the biological role of SPAP may indicate its diagnostic and therapeutic possibilities.

Presence of synaptonemal complex protein 1 transversal filament-like protein in human primary spermatocytes.

The synaptonemal complex (SC) is involved in the pairing of chromosomes during meiosis. We found that antibodies raised against a protein component (P1) of the mouse synaptonemal complex, mouse SCP1, also identified the SC in human primary spermatocytes. Biopsies from 18 men presented with infertility were evaluated by light-field microscopy and grouped into five categories: normal spermatogenesis, Sertoli cell-only syndrome, meiotic disturbances, spermiogenic (i.e. differentiation) disturbances, and other combined disturbances. In all the normal subjects the SCP1 antibody distinctly stained the synaptonemal complexes of primary spermatocytes, whereas Sertoli cells, spermatogonia or spermatids were never stained. In three of the groups, which had germ cells but showed spermatogenic disturbances, the staining was similar to that seen in normal subjects. In sharp contrast to this, in sections from men with Sertoli cell-only syndrome no specific staining was seen. This study demonstrates that a SCP1-related protein is also conserved in the synaptonemal complex in meiotic cells from man. Further studies will reveal to what extent the absence or the non-functionality of SCP1 contributes to male infertility.

Derivatives of 2-nitrofluorene cause changes of human sperm motility.

The effects on human sperm motility characteristics of 2-nitrofluorene and selected derivatives were studied in vitro, using computer aided sperm analysis (Cellsoft). Substances to be tested were dissolved in acetone and added to separated spermatozoa in culture media to final concentrations of 100 and 1000 microM. Aliquots were removed immediately (< 5 min.) and 24 hr after the addition and tested for sperm motility characteristics. Four of the substances tested; 2,4,7-trinitrofluoren-9-one (2,4,7-tNFO), 2,5-diaminofluorene (2,5-dAF), 7-hydroxy-2-nitrofluorene (7-OH-NF) and 2,7-diaminofluorene (2,7-dAF) showed strong detrimental effects on the sperm motility. Slight detrimental effects were also recorded using 2-nitrofluorene and 2,5-dinitrofluorene (2,5-dNF). Weak stimulatory effects were obtained using 2-acetoamidofluorene (AAF) and 2,7-dinitrofluorene (2,7-dNF). No significant effects were seen with 5-hydroxy-2-nitrofluorene (5-OH-NF), 2-aminofluorene (AF), 2-aminofluoren-9-one (AFO), 2-amino-9-hydroxyfluorene (9-OH-AF) or 9-hydroxy-2-nitrofluorene (9-OH-NF). The mechanism behind this effect is not known but it could be speculated that these lipophilic substances interact with the membranes or the cellular respiration.

Purification, structure and partial characterization of the major sperm activating protein complex in human serum.

Serum has long been known to enhance sperm motility, and the major factor that mediates this activation has been shown to be a macromolecule complex with a MW of about 250 kD. This macromolecule, named Sperm Activating Protein (SPAP) has now been purified by a four step protocol. Starting with 100 mL of serum 20 to 200 micrograms of purified SPAP was recovered (N = 40). Studies on the identification and structure of SPAP revealed that the macromolecule is formed by an IgG molecule to which an apolipoprotein A-I molecule is bound. Further studies on the structure of the complex was performed by partial proteolytic cleavage of SPAP by pepsin and papain and interaction with poly- and monoclonal antibodies. Based on these results the most plausible structure of SPAP places one molecule of apo A-I between the two arms of the Fab portion of an IgG4 molecule. Human spermatozoa incubated with polyclonal anti-SPAP serum and a second FITC-labelled antiserum showed fluorescence at a distinct band at the lower part of the sperm head, indicating that SPAP acts by direct interaction with the spermatozoa. The SPAP molecule shows a new mechanism mediated by the immunoglobulins, that may be very important for male fecundity.