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R. A. P. Harrison

Summary. A simple method has been developed for washing spermatozoa: the cell suspension is layered over a solution containing Ficoll and, after centrifugation, the original suspending medium remains as an undiluted layer above the Ficoll solution while the spermatozoa form a loose pellet at the bottom. Removal of the supernatant layers is easily and completely accomplished by aspiration.

When ram spermatozoa were washed by this method, as much as 99·6% of the original medium could be removed during a single washing cycle, while up to 98% of the cells were recovered. Mechanical damage to the cells was minimal and repeatability was high.

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A method has been developed for the simultaneous isolation of spermatozoa and cytoplasmic droplets from bull, boar and ram semen. The glycolytic enzymes, hexokinase (HK), glucose phosphate isomerase (GPI) and lactate dehydrogenase (LDH) were demonstrated in both types of particle: on a per particle basis compared with spermatozoa, droplets contained similar quantities of GPI, less HK and only one-tenth the amount of LDH. The HK was partly bound while the GPI was entirely soluble in both. The LDH was partly bound in spermatozoa but soluble in droplets.

Suspensions of spermatozoa and cytoplasmic droplets were subjected to sudden cooling, freezing or hypo-osmotic shock, and leakage of GPI, HK and LDH into the extracellular medium was measured. Both spermatozoa and droplets released glycolytic enzymes during the treatments, but the droplets were more fragile than the spermatozoa. The proportion of loss varied between enzymes, as well as between droplet and spermatozoon; considerable GPI and little HK was released from both, whereas much LDH was lost from droplets but hardly any from spermatozoa.

The activities of these three glycolytic enzymes were also measured in seminal plasma from bull, boar and ram. The ratios of the activities found were compared with those in droplets or spermatozoa from the same species. It is suggested that much of the glycolytic enzyme activity in seminal plasma arises from disintegrated cytoplasmic droplets rather than from spermatozoa.

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Semen, collected from boars of proven fertility, was concentrated by centrifuging the strained ejaculate at 400 g for 15 min at 20° C and gently resuspending the sperm pellet in one-fifth the original volume of seminal plasma. The concentrated semen was then diluted 1:6 either in a chosen wash medium (see Table 1 for composition of media) and used immediately, or in a milk diluent (MFP), cooled slowly to 5° C over a period of 5 hr, and stored overnight at 5° C.

The spermatozoa were washed by centrifugation at 400 g for 12 min followed by gentle resuspension in a fresh aliquot of the chosen wash medium. Three centrifugations were carried out and samples of the final pellet were resuspended in chosen media. All manipulations of fresh semen were carried out at 20° C, whereas

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A study was made of three methods of washing bull, boar and ram spermatozoa, using as criteria contamination with free-lying cytoplasmic droplets, sperm motility and leakage of glucose-phosphate isomerase and hexokinase into the medium. The methods used were: washing in a Ringer-fructose-phosphate medium, washing in a hypoosmotic phosphate medium and washing with balanced media in the cold after slow cooling and storage overnight at 4° C in a milk diluent. Only by using the last method was it possible to remove adequately the freelying cytoplasmic droplets ; this method also caused least damage to the sperm suspensions.

The problems of washing spermatozoa with respect to cell damage and leakage of enzymes are discussed in the light of the present findings. The evidence suggests that glucose-phosphate isomerase and hexokinase leak from the cytoplasmic droplets as well as from the spermatozoa during washing. Hypo-osmotic media appear to cause an immediate and marked release of these enzymes from the droplets.

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R. A. P. Harrison and S. J. Gaunt

Summary. Two monoclonal anti-sperm hyaluronidase-producing cell lines were isolated following inoculation of mice with ram sperm hyaluronidase monomer. Both lines produced antibodies of the IgG1 class; these bound to ram hyaluronidase after 'Western blotting' but did not recognize the native enzyme. Whereas the IA4 antibody was specific for ram hyaluronidase, and did not react with 'blotted' bull, boar or rabbit hyaluronidase, the 1D6 antibody recognized bull as well as ram hyaluronidase.

The antibodies could be used for immunocytochemical localization of hyaluronidase in fixed spermatozoa. However, although some form of denaturation was required to unmask or form the epitopes with which the antibodies reacted, the degree and type of fixation required was critical, for the epitopes were readily destroyed; in particular, they were very sensitive to chemical modification such as glutaraldehyde treatment.

It could be demonstrated that, like ram, bull spermatozoa contained an extended oligomeric family of hyaluronidase forms, apparently the result of intermolecular disulphide cross-linking of monomers. In spermatozoa of both species, the enzyme was confined to the anterior acrosomal region of the head.

Keywords: hyaluronidase; spermatozoa; bull; ram; monoclonal antibodies

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R. A. P. Harrison and Sally E. Vickers

Summary. Carboxyfluorescein diacetate and propidium iodide were used as fluorescent stains to assess membrane integrity in sperm populations from ram and boar. The living spermatozoa were immobilized with low concentrations of formaldehyde so that individual stained cells could be observed in a suspension with the aid of a fluorescence microscope. Intracellular esterases liberated impermeant-free carboxyfluorescein from the permeant carboxyfluorescein diacetate and caused the product to accumulate and fluoresce green within the acrosome and the mitochondria as well as within the cytoplasm. Most of the spermatozoa (the intact ones) accumulated carboxyfluorescein in all compartments; however, a few cells (those with damaged plasma membranes) accumulated the stain only in the acrosome and/or the mitochondria, while others (all of whose membranes were damaged) remained entirely unstained. The impermeant propidium iodide did not stain any of the (intact) spermatozoa that accumulated carboxyfluorescein throughout their length, but stained all the others (the heads fluoresced red).

The technique appeared to provide more reliable estimations of the percentage of functional cells than did motility estimations or assessments of acrosomal integrity (presence of normal apical ridge). The technique also demonstrated the sensitivity of the sperm plasma membrane to cold shock: virtually all cells rapidly became permeable to the stains after such stress. Assessments of boar sperm samples during preparative incubation for in-vitro fertilization indicated a considerable increase in the percentage of cells with damaged plasma membranes as incubation proceeded, in advance of the increase in the percentage of cells with discharged acrosomes.

Keywords: spermatozoa; fluorescent probes; membranes

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R. A. P. Harrison, J.-E. Fléchon and C. R. Brown

Summary. Acrosin and its zymogen form proacrosin were located in various sperm fractions by biochemical and immunocytochemical techniques, using an anti-acrosin serum that cross-reacts strongly with proacrosin.

If activation of proacrosin was prevented, the zymogen was associated almost entirely with the sperm heads, where it was confined to the anterior segment of the acrosome. Electron microscopy revealed that the acrosomal lumen of such heads remained full of matrix material, and the outer acrosomal membrane remained closely apposed to the other head structures. However, if activation had been allowed to take place, the resultant acrosin and the remaining proacrosin were associated with all sperm fractions and no specific location was observed. In these circumstances, the matrix material was almost entirely absent from the heads, and the outer acrosomal membrane, though usually still present, was only loosely attached.

It is concluded that (1) neither acrosin nor proacrosin are truly membrane-bound; they behave as 'diffusible' or partly soluble proteins, although they display a non-specific affinity for cell membrane and other surfaces; (2) proacrosin is part of the acrosomal matrix material, and is not located specifically on the inner or the outer acrosomal membrane; (3) the matrix material plays an important mechanical role in stabilizing the position of the outer acrosomal membrane relative to the inner acrosomal membrane; it disperses during proacrosin activation.

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Lynn R. Fraser, R. A. P. Harrison and Jane E. Herod

Summary. Earlier studies demonstrated that epididymal mouse spermatozoa have a surface-associated factor which inhibits fertilizing ability in a reversible manner. The factor can be removed from uncapacitated spermatozoa by gentle centrifugation, resulting in immediately highly fertile gametes, and it can be added back to capacitated spermatozoa, resulting in poorly fertile cells in which the acrosome reaction has been blocked. Using such inhibition of in-vitro fertilizing ability as an assay, we have carried out experiments to characterize the factor. It appears to be an anionic polypeptide with M r of approximately 40 000 (according to its behaviour on gel filtration). It is stable to heating at 100°C for 15 min and is not destroyed by proteases at pH 8·0, yet inhibitory activity decreases during sperm incubation in capacitating conditions and is also destroyed in partially purified preparations by endogenous enzyme action during incubation at pH 5·0. Activity is not adsorbed to either concanavalin A–agarose or wheatgerm agglutinin–agarose, suggesting that terminal mannose and N-acetylglucosamine residues are not abundant. The factor causes rapid changes in the patterns of chlortetracycline fluorescence seen on sperm heads, a parameter used to assess the capacitated state. Removal of the factor from uncapacitated cells results in a shift to a predominance of capacitated patterns, while the addition of crude or partially purified factor to capacitated cells inhibits the acrosome reaction and causes a shift to the uncapacitated pattern in acrosome-intact spermatozoa. The factor therefore behaves as a decapacitation factor. However, it appears to differ from other characterized decapacitation factors in terms both of molecular size and of abundance of mannose and N-acetylglucosamine residues.

Keywords: decapacitation factor; capacitation; acrosome reaction; in-vitro fertilization; mouse spermatozoa

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M. T. Kane, M. Norris and R. A. P. Harrison

Summary. The uptake of myo-inositol by preimplantation mouse embryos was investigated using [3H]myo-inositol. Uptake increased about 12-fold between one- and two-cell stages and increased again at the blastocyst stage (> 6-fold compared with the two-cell stage). Uptake at the blastocyst stage was time and temperature dependent; it was stimulated by sodium, inhibited by glucose and appeared to take place mainly via a saturable mechanism. Uptake in the presence of 6·25 mmol inositol l−1 was 1424 fmol inositol per blastocyst per h. About 10% of the [3H]inositol taken up by blastocysts during 8 h in culture was incorporated into lipid. Thin layer chromatography of the lipid showed that most of this inositol was incorporated into lipid material co-migrating with phosphatidylinositol with a small proportion co-migrating with phosphatidylinositol 4-phosphate.

Keywords: mouse; embryo; inositol; phosphoinositides

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R. A. P. Harrison, H. M. Dott and G. C. Foster

Summary. The seminal plasma in sperm suspensions from boar, bull, rabbit, ram and stallion was replaced with simple defined media as completely as possible by a combination of centrifugation through Ficoll and dilution. After this process, motility declined and the cells showed a tendency to agglutinate and/or stick to glass. Varying the ionic strength of the medium had little effect upon these parameters but sperm motility was preserved better in the presence of serum albumin. When a number of purified proteins and other macromolecules were tested individually in this way for their motility-preserving ability, bovine or human serum albumin was consistently the most effective. Defatting the albumin or altering its nature by mild reduction, oxidation or alkylation had little detectable effect on its motility-preserving ability; the protein did not appear to be acting as a chelator of metal ions, for it could not be replaced by EDTA.

The response of the spermatozoa to replacement of seminal plasma varied between species: bull spermatozoa were particularly sensitive and serum albumin had little effect upon their subsequent motility.