Cryopreservation in different concentrations of glycerol alters boar sperm and their membranes.

To test the hypothesis that glycerol would concomitantly affect sperm membrane structure and the function of the intact cells, boar semen (4 ejaculates from 4 boars) was cryopreserved in an egg yolk extender with 0%, 2%, 4%, or 8% glycerol in 0.5-mL straws using previously derived optimal cooling and thawing rates. Increasing glycerol concentrations increased spermatozoal progressive motility immediately after thawing and after 2 hours at 43 degrees C, but decreased the percentage of sperm with normal acrosomal morphology. The mathematical products of the motility and acrosomal integrity scores (MOT x NAR index) were low in 0% and 8% glycerol, and significantly higher in 2% and 4% glycerol. The fluidity of sperm-head plasma membranes, a measure of molecular interaction, was assessed with the lipid probes trans-parinaric acid and cisparinaric acid (tPNA, cPNA), during a 2.5-hour incubation with or without 1 mM Ca2+. Membrane fluidity detected by each probe differed significantly, indicating the presence of at least 2 domains whose constituent molecules had unique dynamics. Behavior of each domain was radically altered by cryopreservation. Increasing glycerol concentration caused a variably faster loss of fluidity in the cPNA domain, and had highly variable effects on fluidity change over time in the tPNA domain. Normal acrosomal ridge (NAR) and the MOT x NAR index correlated significantly with the fluidity of the more mobile cPNA domain (+/- 1 mM Ca2+), supporting the hypothesis of an interrelationship of glycerol concentration during cryopreservation with sperm membrane structure and cell function. The MOT x NAR index may be a useful guide in choosing optimal cryoprotectant concentrations.

Composition and behavior of head membrane lipids of fresh and cryopreserved boar sperm.

Head plasma membranes were isolated from fresh or cryopreserved ejaculated boar spermatozoa and the lipids were extracted for determination of lipid fluidity (n = 6 for fresh and cryopreserved) and for compositional analysis (n = 5 for fresh, 6 for cryopreserved). Composition of the egg yolk extender was also determined. For fluidity determination, the mixed lipids were allowed to form natural liposomes. Bilayer fluidity of these liposomes was analyzed in the presence or the absence of 1 mM Ca2+ with the probes tPNA, which preferentially locates into gel-phase areas, and cPNA, which enters fluid and gel-phase areas equally and thus assesses bulk lipids. Fluidity of liposomes declined significantly during controlled-rate cooling for all samples. Compared to lipids from fresh membranes, gel lipids from cryopreserved cells lost fluidity at a significantly more rapid rate, as did bulk lipids in the presence of Ca2+ (P < 0.001). Fluidity increased during subsequent rewarming (5 to 50 degrees C), again at a slower rate for lipids from fresh cells, with the cryopreservation effect being significant for all probe/Ca2+ combinations (P < or = 0.05). Calcium altered the fluidity characteristics of membrane lipids from fresh but not cryopreserved sperm when analyzed during cooling with cPNA (P < 0.01) and during rewarming with cPNA (P < 0.0001) and tPNA (P < 0.05). Lipids from cryopreserved cells contained significantly less sphingomyelin (14.6 +/- 1.1 vs 22.4 +/- 1.6 mol%) and more phosphatidylcholine (51.5 +/- 2.0 vs 40.5 +/- 2.4%). The octadecanoate (18:0) content in both phosphatidylserine and phosphatidylethanolamine decreased after cryopreservation (P < 0.05). The polyunsaturated fatty acids docosatetraenoate (22:4) and/or arachidonate (20:4) increased in these phospholipids and in sphingomyelin and phosphatidylinositol (P < 0.05). The alterations in the molecular interactions, composition, and Ca2+ sensitivity of membrane lipids may interfere with the normal membrane events of fertilization.

Diet and breed influence the sperm membranes of beef bulls.

Semen was collected from 12 Hereford and 10 Simmental bulls at the conclusion of a 119-day Record of Performance growth trial. Within each breed, the bulls were fed a standard test ration (Diet 1) or an experimental diet consisting entirely of a pelleted concentrate with ground corn cobs as the primary fibre source (Diet 2). Semen was analyzed for motility and morphology while testicular tissue obtained at slaughter the day after semen collection was assessed for seminiferous tubule integrity; none of these parameters varied significantly with breed or diet. The fluidity of head plasma membranes from the spermatozoa was assessed with fluorescence polarization using tPNA. Fluidity decreased over the 160 minute observation period, indicating molecular rearrangments within the head membranes which may reflect sperm changes preceding fertilization. The fluidization displayed a breed-by-diet interaction since membrane fluidity differed significantly between breeds on Diet 1 and between diets for Simmental bulls. Fluidities of some samples were also analyzed with cPNA, and these differed significantly from those obtained with tPNA, indicating the presence of domains in sperm head membranes. Neither diet nor breed affected traditionally measured semen characteristics of Hereford and Simmental bulls, but the membrane dynamics differed between the 2 breeds, and diet affected the sperm membrane dynamics of Simmental bulls.

Treatment of spermagglutination with proteolytic enzymes. I. Sperm motility, vitality, longevity and successful disagglutination.

The treatment of antibody-mediated spermagglutination by corticosteroid therapy has a high incidence of side-effects and sperm washing is often followed by re-agglutination. The possibility of enzymatic disagglutination was therefore investigated. In the first part of the study the effects of four proteases on sperm motility, vitality and longevity were evaluated. Subtilisin had prohibitively detrimental effects even at 10 U/ml. However, chymotrypsin (less than or equal to 500 U/ml), trypsin (less than or equal to 500 U/ml) and papain (less than or equal to 50 U/ml) had no adverse effects. In the second series of experiments one or more of these latter three enzymes was found to disagglutinate spermatozoa which had previously been incubated with sperm-agglutinating antibody-positive sera in 87% of cases. Although further investigation is required, enzymatic disagglutination may be beneficial for the treatment of immunologically mediated spermagglutination.

Combined use of fluorescent peanut agglutinin lectin and Hoechst 33258 to monitor the acrosomal status and vitality of human spermatozoa.

Fluorescein-conjugated peanut agglutinin (PNA) lectin-labelling is an established procedure for assessing the status of the human sperm acrosome. However, unlike the triple-stain technique, PNA-labelling does not provide a simultaneous assessment of cellular vitality. We have therefore evaluated the use of the fluorescent dye Hoechst 33258 (H33258) as a vital stain for use in combination with PNA-labelling. Human sperm populations were stained for 1 min with 1 microgram/ml H33258 in culture medium and then washed through 2.0 (w/v) polyvinylpyrollidone columns and air-dried onto microscope slides. H33258 was found to provide vitality assessments comparable to those obtained using the standard eosin-exclusion method. However, best results were obtained with an ethanol fixation step between air-drying and PNA-labelling. This vitality assessment was found to be more reliable than that provided by Trypan blue staining under conditions equivalent to the triple-stain technique. There was no alteration of PNA-labelling due to the H33258 although ethanol fixation actually provided more uniform PNA-labelling than previously obtained without ethanol fixation. Consequently, we have stopped using the triple-stain technique for assessing human sperm acrosome reactions and now use the H33258/PNA procedure routinely.

The spontaneous acrosome reaction of human spermatozoa incubated in vitro.

Motile human sperm populations were prepared from liquefied semen (10 donors x 3 replicates) using Percoll density gradients at 30-60 min post-ejaculation. Sperm suspensions were incubated in a complex 'synthetic tubal fluid' culture medium (STF) at 37 degrees C under 5% CO2 in air for up to 36 h. Parallel aliquots were incubated with 50 microM A23187 to induce maximum acrosome loss (ARMAX). Acrosome reactions were assessed using both the triple-stain (TS) technique and fluorescent peanut agglutinin (PNA) lectin-labelling. During incubation, the proportion of TS acrosome reacted spermatozoa increased from 9.1 to 54.3% with ARMAX being 68.3%. Spermatozoa showing intact acrosomes by PNA labelling decreased from 68.4 to 26.1% over 36 h of incubation (ARMAX = 13.8%). Simultaneously, spermatozoa showing complete acrosomal loss (no PNA labelling) increased from 8.1 to 27.0% (ARMAX = 46.3%). Therefore, while only 23.5% of cells were actually undergoing acrosomal changes at the start of incubation, this had increased to 46.9% after 36 h (ARMAX = 40.7%). These experiments clearly show that even in selected populations, not all human spermatozoa are capable of undergoing an acrosome reaction. However, the incidence of acrosomal changes after 36 h of incubation did approach the ARMAX. These levels of spontaneous occurrence of the human sperm acrosome reaction were higher than those reported in many other in-vitro incubation studies: an improvement that may be attributable to the more physiological nature of the STF culture medium.

Calcium dependence of human sperm fertilizing ability.

The Ca2+ dependency of human-sperm fertilizing ability was investigated using a modified Tyrode's medium either containing 2.4 mM CaCl2 (CA medium) or with the CaCl2 replaced by SrCl2 and 0.1 mM EGTA added to chelate any residual Ca2+ ions (SREG-medium). Ten washed sperm populations incubated in either medium for 0, 6, and 22 hours showed the same occurrence of acrosome reactions (by fluorescent lectin labelling and triple stain). A further 3-hour incubation after washing into fresh CA medium resulted in only a slight increase in acrosome reactions in both media. Eight sperm populations preincubated overnight in CA and SREG media were coincubated for 1 hour with previously salt-stored human zonae pellucidae also in the same media. Significantly more motile spermatozoa were bound to more of the zonae in CA medium (53.9% vs. 27.6% of zonae with 13.8 vs. 4.3 sperm/bound zona). In three hamster egg penetration test (HEPT) experiments, sperm populations preincubated overnight in either CA or SREG media were coincubated with hamster oocytes prepared in the same media. Only 2.1% of oocytes (1.0 polyspermy) were penetrated in SREG medium, cf., 30.9% of oocytes (1.3 polyspermy) in CA medium. These results demonstrate that while Sr2+ ions can substitute fully for Ca2+ in the capacitation and acrosome reaction of human spermatozoa, sperm-zona, and sperm-oolemma interaction seem to involve some more Ca2+-specific process(es). Furthermore, the increased HEPT fertilizing ability of human spermatozoa using overnight preincubation in SREG medium and CA medium for the test cannot be explained on the basis of differential kinetics of capacitation or the acrosome reaction.

Semi-automated analysis of manually reconstructed tracks of progressively motile human spermatozoa.

Several alternative algorithms for computer-assisted derivation of measurements of movement characteristics from manually reconstructed tracks of progressively motile human spermatozoa were compared. Fifty tracks were reconstructed at 30 Hz from video recordings and analysed using traditional manual methods and by four combinations of computer algorithms. The best algorithm set was identified ('Videomot.mdpt') and the values for the curvilinear, average path and linear velocities (VCVL, VAVE and VLIN respectively), the amplitude of lateral displacement of the sperm head about the axis of progression (AH) and the number of times the sperm head crossed the average path (the 'beat/cross frequency', BXF) obtained using it were compared to those obtained by manual analysis. There was a considerable time saving when the computer-assisted method was used and the values it gave for the various movement characteristics showed good correspondence with those obtained manually. In addition, repeated data entry and analysis was found to be highly reproducible. When the tracks were re-plotted at 6 Hz (as used by the multiple-exposure photomicrography method for sperm motility analysis) insufficient information remained in the tracks for reliable determination of anything other than VLIN. We conclude that the Videomot.mdpt program provides reliable values for the movement characteristics of progressively motile human spermatozoa, and believe it will be of great value in the validation of commercial systems providing automated sperm movement analysis and in laboratories which do not have access to such costly equipment.

Specific labelling by peanut agglutinin of the outer acrosomal membrane of the human spermatozoon.

Experiments to bind fluorescein-conjugated Arachis hypogea (peanut) agglutinin (FITC-PNA) to washed human spermatozoa demonstrated that this lectin binds to the acrosome region in air-dried preparations. Since there was no binding when labelling was performed in suspension, and comparable labelling to that seen in air-dried preparations was seen when spermatozoa treated with saponin (to lyse the plasma membrane) were labelled in suspension, the lectin must bind to an intracellular structure, probably the outer acrosomal membrane. This was confirmed by ultrastructural localization of colloidal gold-conjugated lectin in saponin-treated spermatozoa. Treatment of spermatozoa with the detergent Nonidet P-40 caused a marked change in the binding pattern: more spermatozoa showed binding in the equatorial segment of the acrosome with no binding in the anterior cap region. A comparable, less marked, change was seen when spermatozoa were incubated overnight under conditions known to support the capacitation and spontaneous acrosome reactions. Treatment with the calcium ionophore A23187 for 1 h to induce acrosome reactions artificially in uncapacitated spermatozoa resulted in the appearance of patchy acrosome fluorescence. From these experiments it is concluded that PNA binds specifically to the outer acrosomal membrane, and that FITC-PNA-labelling may be used to monitor the human sperm acrosome reaction.

The use of strontium-substituted media for capacitating human spermatozoa: an improved sperm preparation method for the zona-free hamster egg penetration test.

An improved procedure for human sperm capacitation based upon calcium deprivation using a modified Tyrode's medium is described. Replacement of CaCl2 by SrCl2 significantly increased the penetration of zona-free hamster eggs after 20 hours of sperm preincubation, compared with parallel sperm aliquots incubated in normal medium. The effect of an extra 4 hours' sperm preincubation produced no significant shift in fertilizing ability. Addition of 0.1 mM ethyleneglycol-bis-(beta-amino-ethyl ether)-N,N,N',N'- tetraacetic acid (EGTA) to the strontium-substituted medium resulted in a further significant increase in the penetration rate as well as a significant increase in polyspermy. The average increase in the penetration capacity (penetration rate X polyspermy) of the strontium/EGTA-preincubated spermatozoa was approximately tenfold, with no significant shift as a result of an extra 4 hours' preincubation.