CHEMICAL COMPOSITION OF THE ACROSOMES OF RAM SPERMATOZOA E. F. HARTREE and P. N. SRIVASTAVA A.R.C. Unit of Reproductive Physiology and Biochemistry, 307 Huntingdon Road, Cambridge {Received 27th May 1964) Summary. Material extracted from ram spermatozoa with 0\m=.\0125 n-NaOH was separated into lipid and glycoprotein fractions. By use of an anionic detergent (Hyamine 2389) acrosomes were separated from ram spermatozoa and also fractionated into lipid and glycoprotein. The chemical compositions of the two glycoprotein fractions, as well as of the two lipid fractions, show marked similarities. Taking into consideration the chemical changes that may occur during the isolation of these frac- tions it is deduced that they provide a reasonable approximation to the composition of the acrosomes. Ofthe amino acids glutamic acid predom- inates. The following sugars are present: mannose, galactose, fucose, glucosamine, galactosamine and sialic acid. The residue left after extract- ing ram spermatozoa with n-NaOH contains polysaccharide of which the constituent sugars are glucose, galactose and mannose. All the sialic acid of ram spermatozoa appears to be contained in the acrosome. The sialic acid content of the spermatozoa of other species has been measured and the possible role of sialic acid in sperm physiology is discussed. INTRODUCTION Clermont, Glegg & Leblond (1955) found that the component of the acrosome which is stained in the periodic acid-Schiff (pas) reaction could be removed from guinea-pig spermatozoa by extraction with 0-1 N-NaOH. Hathaway & Hartree (1963) showed that acrosomes can be removed from washed ram spermatozoa as effectively by 0-01 N-NaOH as by 0-1 N-NaOH; within that range of concentrations there was very little variation in the quantities of nitrogen and of orcinol-reactive sugar that passed into the alkaline extracts. This was taken as evidence that a discrete unit, the acrosome, was being dissolved. The possibility remained that material other than that present in the acrosome was simultaneously dissolved from the sperm cell in quantities which were also independent of the concentration of alkali. It was shown in the same paper that treatment of ram spermatozoa with hexadecyltrimethylammonium bromide (cetyltrimethylammonium bromide, ctab) caused membranous material, and in some cases apparently intact acrosomes, to be removed from the sperm head. Such detached elements, like the acrosomes of intact sperma¬ tozoa, were strongly stained by the pas and Giemsa techniques. 47 Downloaded from Bioscientifica.com at 09/30/2021 08:48:50AM via free access 48 E. F. Hartree and . Srivastava We have now examined the effects ofother detergents upon ram spermatozoa. We have improved the methods for detachment of morphologically intact acrosomes and have been able to obtain them as suspensions which are virtually free from spermatozoa. The chemical composition of such acrosomal prepara¬ tions, and that of the material extracted from spermatozoa by alkali, has been determined. The sialic acid content of the semen of a few other species has also been measured. MATERIALS AND METHODS Semen was collected twice weekly from a group of about twenty rams, and pooled. The spermatozoa were separated from the seminal plasma within 1 hr of collection and were washed with calcium-free Ringer solution as described by Hathaway & Hartree (1963). The final suspension was made up in Ringer solution to twice the original semen volume. Spermatozoa were stained with Giemsa solution as described by Hancock (1952) except that preparations were not fixed and the slides were left in the stain solution for 16 hr at 20° C. Bull and rabbit semen were dealt with in the same way. Epididymal bull semen was provided by Dr H. M. Dott and cock semen by Dr C. Polge. DETACHMENT OF ACROSOMES BY DETERGENTS A suspension of washed ram spermatozoa in Ringer solution was centrifuged and the cells were made up to the same volume with 0-9% sodium chloride (saline). Portions of this suspension were mixed with equal volumes of solutions of the detergents in saline and were incubated at 37° C for 45 min. Observations made on stained preparations of spermatozoa treated by such methods are given in Table 1. Teepol XL and ctab removed the great majority of acro¬ somes, but both acrosomes and tails showed serious damage. Manoxol OT and Hyamine 2389 gave better results : the former was more effective in removing acrosomes but the latter caused less damage to the tails (PI. 1, Fig. 1). When the Hyamine-containing suspension was centrifuged for 15 min at 1500 g the super¬ natant fluid contained shrunken but characteristically stained acrosomes (PI. 1, Fig. 2). Further experiments with Hyamine led to a procedure by which 70 to 90% of acrosomes could be detached without appreciable damage to the tails. This is described below (B). ISOLATION OF ACROSOMAL GLYCOPROTEIN FRACTIONS A. Isolation from an alkaline extract of spermatozoa A mixture of 40 ml of washed spermatozoa and 40 ml of 0-025 N-NaOH, having a pH of 11-4, was held at 37° C for 45 min and centrifuged at 6000g for 15 min. The almost clear supernatant fluid (75 ml) was brought to pH 6-5 with acetic acid and could be stored at —20° C if necessary. Each extract was mixed with four volumes of cold (—20° C) acetone, and centrifuged at 8000 g and 5° C for 10 min. The white sediment was suspended in about 10 ml of water and dialysed overnight at 5° C against 51. of distilled water. Lipid was removed from the dialysed solution by the procedure of Hartree & Mann (1961). The resulting precipitate was washed with chloroform and air-dried. This material Downloaded from Bioscientifica.com at 09/30/2021 08:48:50AM via free access PLATE 1 Magnification of spermatozoa: x920 in Figs. 1 and 2; x2200 in Figs. 3 and 4. All preparations were stained by the Giemsa method. Fig. 1. Ram spermatozoa treated with 0-025% Hyamine for 45 min at 37° C. Fig. 2. Shrunken acrosomes separated from suspension shown in Fig. 1. Fig. 3. As Fig. 1, using 0-05% Hyamine for 90 min. Fig. 4. Acrosomes separated from suspension shown in Fig. 3. (Facing p. 48) Downloaded from Bioscientifica.com at 09/30/2021 08:48:50AM via free access Chemical composition of ram acrosomes -a -o u 3 U S ( g •s 3|w S -S >s_bp ~3 "•¿ -a •S S c a S -a 5 c '> ta c o. OT3 " til 8" « ·ß.|c b s S s n u 1 ^ 5 ^ U d o « g a a-0 ¬ m3 ' ¡ OC S Su - 0 S « S c C u %r, sO ìn < ci rt SU tQ 9 »2 -û g o <3* T3 rt S '3 ei ^_Q - s« s P "e 9 S3 o. J3 "5 "3 ¬ I «fe ñ ü G M -à c c« M u c [ -t-» & 2- pu- .s « ¿--5^ •a-i?.? 3 S « * ; í S _. r! .a £ u 0 « 1 _, tZS iJJ O œ «- f-( "_ o a cm >,O.s .2* 3 V "o S m s -. o"S, -1 g-s 3 ai < Cß e H .rt t/j ss s 3 U Downloaded from Bioscientifica.com at 09/30/2021 08:48:50AM via free access 50 E. F. Hartree and . Srivastava will be referred to as glycoprotein A. The extracted lipid was obtained as a solution in chloroform-methanol. This was shaken with 0-2 vol of 0-01 M-MgCl2 to remove impurities and the lower layer was evaporated in a rotary evaporator. The lipid residue was stored in chloroform solution at —20° C (lipid A). The supernatant fluid from which the white sediment had been separated was concentrated to small bulk in a rotary evaporator and treated with an equal volume of 10% trichloroacetic acid. The solution remained clear and it was therefore assumed that all the protein had been precipitated by acetone. -Washed ram spermatozoa pH 11.4, Hyamine pH 6-1, centrifuge centrifuge I Supernatant Sedimenl Sediment solution (discarded) resuspend, acetcne at centrifuge 5°C, centrifuge Sediment Supernatant suspen¬ (discarded ) sion of acrosomes Supernatant Sediment solution Upoglycoprotein ethanci, (discarded) centrifuge chLoroform methanol - Supernatant Sediment Solution of Insoluble solution LIPID A residue (discarded) GLYCOPROTEIN A chloroform methanol Solution of Insoluble LIPID residue GLYCOPROTEIN Flow sheets for separation of lipids and glycoproteins. B. Isolation from detached acrosomes A suspension of washed ram spermatozoa (40 ml) in saline was incubated with an equal volume of 0-1% Hyamine, in 0-067 M-phosphate buffer pH 6-1, at 37° C for 90 min. At this stage some of the detached acrosomes were distorted but still clearly recognizable (PI. 1, Fig. 3). The suspension was centrifuged for 15 min at 1500g, the sediment was resuspended in fresh saline and centri¬ fuged again. The combined supernatant fluids contained acrosomes with very few (<1%) spermatozoa and sperm tails (PI. 1, Fig. 4). The sediment contained spermatozoa, of which about 80% were without acrosomes, and free acrosomes equivalent to about 20% of the spermatozoa. Thus the supernatant fluid con¬ tained in suspension approximately 60% of the acrosomes in the original sperm suspension. The supernatant fluid was treated with an equal volume of absolute ethanol, left overnight at 4° C, and centrifuged. The precipitate was resuspended in 7-5 ml ofwater and lipids were removed as described above. The final products were the air-dried glycoprotein and a solution oflipids in chloroform. These will Downloaded from Bioscientifica.com at 09/30/2021 08:48:50AM via free access Chemical composition of ram acrosomes 51 be referred to as glycoprotein and lipid respectively. When the lipid solutions were required for analysis, phosphate buffer was replaced by saline as a solvent for Hyamine.