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Summary. Bound sialic acids on rat spermatozoa were assayed by oxidation with 1 mMNaIO4 at 0\ s=deg\ C, liberating C-9 as formaldehyde which was further quantitated using 3 \ x = r e q -\ methyl-2-benzothiazolinone. The mean \ m=+-\ s.d. (n = 20) content of bound sialic acids of spermatozoa from the caput and cauda epididymidis was 50\m=.\9 \m=+-\ 8\m=.\0and 25\m=.\2 \m=+-\ 3\ m=. \ 8 nmol/108 spermatozoa respectively. About 85% of the former and 75% of the latter could be extracted by 1% Triton X-100 and 2 mM-dithiothreitol. About 70% of the former and 20% of the latter were released by neuraminidase from Vibrio cholerae. About 40% of the former and 30% of the latter were sensitive to trypsin. During sperm maturation, the decrease in the total bound sialic acids was due to the decrease in the neuraminidase-sensitive but not the neuraminidase-resistant sialic acids.

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Non-motile spermatozoa freshly extruded from the rat caudal epididymis can be initiated to full motility immediately after diluting with 0.9% NaCl. The motility initiation was dependent on the pH, viscosity and osmolality of diluent. Diluent with pH 4 to 8 could optimally initiate the motility. Osmolality of most diluents suitable for the initiation was between 130 to 600 mOsm/kg. The motility initiation was inhibited by Hg2+ greater than Cu2+ greater than Cd2+, chlorpromazine, Triton X-100 and SDS. The following compounds showed essentially no inhibitory effect: EGTA, chlortetracycline, calcein, ruthenium red, phloridzin, myo-inositol and carnitine. The findings suggested that spermatozoa were kept in quiescence in the cauda epididymis not by the pH, osmolality, viscosity, myo-inositol, carnitine, Ca2+ or K+ of the caudal epididymal fluid. It was also suggested that motility initiation did not involve Ca2+, calmodulin and transport of Ca2+ or glucose across sperm membrane.

Spermatozoa from rat epididymis were incubated with [32P] orthophosphate and the radioactively labeled proteins were solubilized for analysis by electrophoresis in SDS-gels or in two-dimensional gels by isoelectric focusing and SDS electrophoresis. Three major phosphorylated protein bands of Mr 42,700, 56,200, and 76,200 were identified together with several minor phosphorylated proteins. The phosphorylated proteins of Mr 42,700 and 76,200 were more heterogeneous in charge than the one of Mr 56,000. The major phosphorylated proteins were not found in the isolated heads of cytosol derived from sperm sonicate. They were not solubilized by 1% Triton X-100 and 2 mM DTT, which removed the plasma membrane and mitochondria, but they were solubilized by 6 M urea and 5 mM DTT away from the insoluble fibrous sheath which contained no appreciable radioactivity. Most of the major phosphorylated bands were solubilized by 2% SDS and 4 mM DTT, leaving the insoluble outer dense fiber-connecting piece (ODF-CP) complex with some of the proteins. The ODF-CP complex of the spermatozoa from the cauda epididymis contained more of the major phosphorylated bands than did that of the spermatozoa from the caput region. Treatment with 1% SDS alone can solubilize about half of the major phosphorylated bands from the spermatozoa of the caput region and essentially none from the spermatozoa of the caudal part. The latter required 1% SDS and 13 mM DTT to achieve solubilization, suggesting the formation of disulfide bonds holding the three major phosphorylated proteins to some intracellular structure during sperm maturation.

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