No abstract

The conformation of 24p3 protein purified from mouse uterine luminal fluid was studied by circular dichroism spectroscopy in 200–300 nm. At pH 7.4, the spectrum in the UV region appears as one negative band with a minimum mean residue ellipticity of ‐3,600 deg.cm2.dmole−1 at 217 nm, suggesting a very low or no helical content, but a considerable amount of β‐form, β‐turn, and unordered form in the protein molecule. This agrees with the predicted secondary structures consisting of only one a‐helical segment of residues 150–163 and nine segments of residues 28–35, 50–60, 67–72, 78–86, 94–97, 106–114, 119–125, 136–140 and 166–172 in β‐forms, which would construct two orthonormal β‐sheets to form a less polar β‐barrel. The environments around Trp‐31 and Trp‐81 of this protein were studied by intrinsic fluorescence and solute quenching. They give an emission peak at 332 nm, and only about 21% of them are accessible to quenching by acrylamide. This together with their low accessibility to either CsCl or KI suggests that they are located in the less polar P‐barrel. Hydrophobic compounds such as fatty acids, retinoids, and cholesteryl oleate in the protein solution diminish the protein fluorescence. Analysis of the fluorescence data suggests that the protein has a binding site for hydrophobic ligand. The association constants for the complex formation are 1.03 × 106M−1, 1.92 × 105M−1, 2.38 × 105M−1 or 1.25 × 105M−1for cholesteryl oleate, oleic acid, retinol, or retinoic acid at pH 7.4. Analysis of the equilibrium binding data from binding assay using [H3]‐retinol and [H3]‐retinoic acid reveals a singular type of retinoid‐binding site in the protein with the association constant of 4.92 × 105M−1 and 1.17 × 105M−1 for retinol and retinoic acid, respectively. Trp‐31 or/ and Trp‐81 is in or very near the binding site and the gross conformation of protein changes considerably as the formation of protein‐ligand complex.

Summary The reproductive‐derived serine protease inhibitor Kazal‐type (Spink) has been identified in seminal plasma, and Spink–spermatozoa binding has been illustrated in many mammalian species including human. We used mice as experimental animal to study the mode of Spink action in the modulation of mammalian sperm activity. A Spink3‐binding zone was cytochemically stained on the sperm head at apical hook separated from intact acrosome, whether the cells were capacitated or not. The Spink3–spermatozoa binding neither changed the population of cells in the uncapacitated, capacitated and acrosome‐reacted status nor affected the capacitation‐related protein phosphorylation and cell motility enhancement. Despite that, the Spink–spermatozoa interaction resulted in decreasing the intracellular calcium concentration ([Ca2+]i) of the cell head and suppressing both the acrosome reaction induced by Ca+2 ionophore A23187 and the cell fertility. Furthermore, Spink3 seen on the head of spermatozoa in the uterine cavity after coitus could be removed by the trypsin‐like activity in the uterine fluid of oestrous females, and free Spink3 in the uterine cavity suppressed the protease activity. We integrated our data to shed light on the molecular mechanism of how Spink and its inhibiting protease are interplayed to modulate the activity of mammalian spermatozoa during their transit in the reproductive tract.