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E. F. HARTREE

A.R.C. Unit of Reproductive Physiology and Biochemistry, 307 Huntingdon Road, Cambridge CB3 0JQ

(Received 21st November 1974)

Using histochemical techniques Mancini et al. (1964) established that hyaluronidase, a lysosomal enzyme, is localized in the acrosomes of bull spermatozoa. Acid phosphatase, the customary histochemical marker for lysosomes, is also concentrated mainly in the acrosomal regions of intact spermatozoa of several animal species (see papers quoted by Allison & Hartree, 1970). The finding of hyaluronidase in a suspension of ram sperm acrosomes (Srivastava, Adams & Hartree, 1965), and later of a number of other typical lysosomal hydrolases, including acid phosphatase (Allison & Hartree, 1970), led the latter authors to suggest that the acrosome is a specialized lysosome that has evolved to facilitate, by enzyme action, the sequential steps of penetration of the spermatozoon through the egg investments. Additional evidence for the lysosomal nature of the acrosome is provided by the analogous histochemical

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E. F. HARTREE and T. MANN

The earliest investigations on the lipids of spermatozoa are those of Miescher (1878) who reported that the ether-extractable material in the spermatozoa of bull and salmon was about 50 % lecithin. At that time, of course, the term lecithin had no precise chemical connotation. However, a high concentration of phospholipid was subsequently shown to be a general characteristic of spermatozoa where it is present mainly in the mid-pieces and tails. The first inquiries into possible metabolic roles for the phospholipids of spermatozoa were made by Lardy & Phillips (1941 a, b). These authors compared the motility of bull spermatozoa in whole semen with that of spermatozoa which had been separated from the seminal plasma and resuspended in Ringer's solution. Redenz had already shown in 1933 that the motility of bull spermatozoa in whole semen is actively maintained under both aerobic and anaerobic conditions. This is possible because in whole semen
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A. C. ALLISON and E. F. HARTREE

Summary.

Ram spermatozoa were washed with hypotonic tris-HCl buffer and obtained almost free of seminal plasma and cytoplasmic droplets. Acrosomes were dislodged by incubating the spermatozoa with a cationic detergent (Hyamine 2389). The acrosomal preparation and the buffer washings were examined for the following lysosomal enzymes: acid phosphatase, aryl sulphatase, β-N-acetylglucosaminidase, phospholipase A and proteases. All enzyme activities were detected, both in washings and in acrosomal preparations. The levels of activity in the latter were much higher than could be expected on the assumption that all activities were due to contamination by washings. Protease activity was greatest at pH 7·5.

After vital staining with Euchrysine 3R, acrosomal fluorescence in ram, bull, boar and human spermatozoa is not very conspicuous. However, acrosomes of guinea-pig, hamster and several rodents show the brilliant orange-red fluorescence typical of lysosomes. As spermatids mature, red-fluorescing granules around the Golgi zone condense to form the red-fluorescent pro-acrosomes.

Acid phosphatase was detectable histochemically in granules, pro-acrosomes, acrosomes and the pellets obtained by high-speed centrifugation of acrosomal preparations. At all developmental stages, histochemical tests for bromochloroindoxyl acetate esterase showed that this enzyme was present only in the acrosome.

The evidence suggests that the acrosome is a specialized lysosome which evolved to facilitate fertilization in multicellular organisms.

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C. R. Brown and E. F. Hartree

A.R.C. Unit of Reproductive Physiology and Biochemistry, University of Cambridge, U.K.

The gelatin-film test for neutral proteinase activity (Gaddum & Blandau, 1970) has been applied to rodent spermatozoa in several laboratories (for details see Brown & Hartree, 1976). There is general agreement that activities are low in rat and mouse spermatozoa. More recently Erickson & Martin (1974) have concluded that the neutral proteinase in mouse spermatozoa is not acrosin. They used a sensitive trypsin assay based on the release of [3H]methanol from tosylarginine [3H]methyl ester and found that the very low hydrolytic activities in fractions from mouse spermatozoa were not decreased by inhibitors of acrosin. As far as we are aware this is the first report of failure to detect acrosin in mammalian spermatozoa. Since this finding is contrary to our experience we are giving evidence for the presence of acrosin in mouse spermatozoa.

Fractionation of spermatozoa

Epididymides

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C. R. BROWN and E. F. HARTREE

The trypsin-like proteinase (TLP) of rabbit spermatozoa (Stambaugh & Buckley, 1969) attracted interest following the discovery that trypsin inhibitors impede the penetration of spermatozoa through the zona pellucida (Stambaugh, Brackett & Mastroianni, 1969; Zaneveld, Robertson & Williams, 1970). Stambaugh & Buckley (1969) recovered most of this proteinase in an acrosomal fraction prepared by treating spermatozoa with the surfactant Hyamine 2389 (Hartree & Srivastava, 1965). Using fluorescein-labelled trypsin inhibitors, they established for several mammalian species that TLP is restricted to the acrosomal region of the sperm cell (Stambaugh & Buckley, 1970).

Difficulties arise when attempts are made to correlate this work with electron micrographic studies of the changes undergone by rabbit and rodent spermatozoa during egg penetration and with electron micrographs of spermatozoa treated with Hyamine. At the stage when a spermatozoon enters the

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R. R. HATHAWAY and E. F. HARTREE

Summary.

Various procedures have been used to extract or detach the acrosomes from ram and bull spermatozoa. Spermatozoa from pooled ram ejaculates, or individual bull ejaculates, were first washed free of the seminal plasma, suspended in Ringer's solution, and then incubated at 37° C with solutions of sodium hydroxide, sodium carbonate, or cetyltrimethylammonium bromide (ctab). An alternate procedure consisted of shaking the Ringer's suspension of spermatozoa with small glass beads. Staining of spermatozoa by the Giemsa or pas methods showed that the acrosomes were apparently dissolved by 0·0125 n-NaOH, and that they were detached as such by 1 ·5 mm ctab or by shaking with glass beads. Nitrogen and sugar determinations on extracts from spermatozoa confirmed the removal of materials from the cells. Because most of the extractable sugar was released in 0·0125 n-NaOH, it is suggested that a structural element, such as the acrosome, was being detached. Release of hyaluronidase from the treated spermatozoa was also observed. Ram sperm extracts contained four antigens, all of which were similar to antigens in ram seminal plasma, as revealed in double-diffusion agar plates. Antisera also had sperm agglutination titres up to 81.

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E. F. HARTREE and P. N. SRIVASTAVA

Summary.

Material extracted from ram spermatozoa with 0·0125 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 fractions it is deduced that they provide a reasonable approximation to the composition of the acrosomes. Of the amino acids glutamic acid predominates. The following sugars are present: mannose, galactose, fucose, glucosamine, galactosamine and sialic acid. The residue left after extracting 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.

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C. R. Brown and E. F. Hartree

Summary.

Cock spermatozoa, like trypsin, induced a rapid fall in the viscosity of gelatin solutions but ram spermatozoa and inhibitor-free ram acrosin were ineffective. The gelatin-hydrolysing activity in cock spermatozoa was solubilized at pH 8 in the presence of calcium ions but comparable extracts of ram spermatozoa were inactive. Both extracts showed acrosin activity (assayed with benzoylarginine ethyl ester). The two catalytic activities of cock spermatozoa were each susceptible to the same trypsin inhibitors and during fractionations they were not separable. We deduce that cock acrosin, and probably some other avian acrosins, have the power to degrade dissolved gelatin while ram acrosin does not. The acrosin in cock spermatozoa, unlike that in ram spermatozoa, was inactivated at pH 2·7. Acid extracts of the former contain an inactive precursor of acrosin which undergoes spontaneous re-activation in buffers, pH 8, containing calcium ions. In this respect it resembles the proacrosin of rabbit testis.

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P. N. SRIVASTAVA, C. E. ADAMS and E. F. HARTREE

Summary.

Cell-free enzyme preparations consisting of lipoglycoprotein were obtained from acrosomes of ram, bull and rabbit spermatozoa. The ram and bull preparations showed proteolytic and hyaluronidase activities. Preparations from the three species brought about dispersal of the cumulus oophorus and corona radiata of newly ovulated rabbit eggs. In some cases the zona pellucida was also removed. Iodoacetate and a polyanionic hyaluronidase inhibitor each reduced the denuding activity of the ram preparation and to a lesser extent of the bull preparation. Heating to 100° C also reduced the activity of the ram and bull preparations. It is concluded that proteolytic enzymes in spermatozoa contribute to the denudation of rabbit eggs and probably facilitate penetration of the zona pellucida.

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R. B. L. Gwatkin, L. Wudl, E. F. Hartree and E. Fink

Incubation of hamster eggs with trypsin or chymotrypsin, but not with a series of glycosidases or lipases, prevents capacitated spermatozoa from binding to the zona pellucida so that fertilization is prevented (Hartmann & Gwatkin, 1971; Gwatkin, Williams & Andersen, 1973). Inactivation of the binding sites for spermatozoa on the zona pellucida by a trypsin-like protease released by discharge of cortical granules also appears to be responsible for the zona reaction which prevents polyspermy (Gwatkin, Williams, Hartmann & Kniazuk, 1973). Acrosin is also a trysin-like protease and it can be extracted from sperm acrosomes. We therefore investigated whether the fertilization of hamster eggs is blocked by incubation with soluble acrosin.