Sialic acid was estimated both chemically in populations of heads and tails of spermatozoa from normospermic human seminal fluid and by transmission electron microscopy, using a specific labeling technique. The results of both procedures show that sialic acid is located exclusively in the head plasma membrane and in the acrosomal membranes. It is suggested that localization of sialic acid close to that reported for neuraminidase in sperm might be of importance in the fertilization process.Key Words: Spermatozoa-human; Sialic acid-localization in sperm; Spermatozoa-ultrastructure.
INTRODUCTIONIn a previous paper [31 we examined sialic acid contents in human spermatozoa of specimens varying in seminal fluid quality. The significance of the observed differences between normospermic and oligozoospermic samples was discussed in respect to the possible role of this compound in the process of fertilization.The assessment of sialic acid distribution among different parts of the spermatozoon might contribute to a better understanding of the involvement of sialic acid in sperm function. The present study was aimed at the chemical determination of sialic acid in populations of heads and tails from normospermic origin separately and at its ultrastructural localization in the intact cell by transmission electron microscopy.
MATERIALS AND METHODSThirteen normospermic seminal fluids (62-216 X 10' cells/ml) with motilities above 50% and motility grades ranging from 2.0 to 3.5 [l] were used. Isolation and cleavage of spermatozoa were performed by a modification of the procedure reported by Witkin [I21 briefly as follows: seminal fluid, diluted with 0.01 M Tris-HCI, 0.5 M NaC1, 1 mM EDTA, pH 7.5, were centrifuged at 3500 rpm for 10 min at 0-4°C and the sediment washed three times. The final pellet was suspended in 0.05 M Tris-HCI buffer, pH 7.5, sperm density was reestimated and sodium lauroyl sarconisate (Sarcosyl, Sigma), which decapitates the mammalian sperm, was added to a final concentration of 1.5%. The suspension was stirred at 37°C for 25 min and sonicated for 90 sec at maximal speed, using a cell disruptor, Model W-370 (Heat Systems, Ultraconics Inc.). Three fractions were separated by centrifugation at 500 rpm for 10 min at room temperature: ( I ) the (Fluka), and thin sections were cut with a LKB ultratome 111. The preparations were examined using a Jeol 100 C electron microscope.
RESULTS AND DISCUSSIONSialic acid is present exclusively in the sperm heads. Minute amounts in tails, if present, would be lower than the sensitivity limit of the method of determination when dealing with protein amounts up to 0.35 mg. Mean amount of sialic acid in heads was 20.1 -c 3.2 (SE) ygil mg protein and comparison of this value to that reported by us [3] in whole spermatoza of normospermic semen (6.5 pg sialic acid per lo8 sperm cells) which, by appropriate calculation 191 would correspond to 3.2 pg/l mg protein, is compatible with a massive localization of sialic acid residues in the heads. These results are in agreement with data ...