Acrosome-intact Cells Research Articles

Cryopreservation of rabbit spermatozoa using acetamide in combination with trehalose and methyl cellulose

Glycerol is not an effective cryoprotectant for rabbit spermatozoa; therefore, rabbit spermatozoa were used as a model for developing cryopreservation procedures for other cell types which also freeze poorly when glycerol is used as the cryoprotectant. Experiments were conducted to 1) compare several published protocols for cryopreserving rabbit spermatozoa; 2) determine if removal of seminal granules, required for flow cytometry analysis, affects the motility of rabbit spermatozoa; and 3) determine if using a combination of cell permeating cryoprotectants (acetamide) with cell nonpermeating cryoprotectants (trehalose and methyl cellulose; MC), can increase the recovery of viable rabbit spermatozoa after cryopreservation. Media containing acetamide as a cryoprotectant were found to be most effective for rabbit spermatozoa. The cryoprotectants ethylene glycol, dimethylsulfoxide and glycerol were not effective for cryopreserving rabbit spermatozoa. Second, rabbit spermatozoa could be centrifuged through a Percoll gradient composed of equal volumes of Prcoll and a HEPES-buffered sperm medium. This centrifugation removed all seminal granules without affecting the percentage of motile spermatozoa after initial sperm dilution (85 vs 74%) or after cryopreservation (35 vs 30%), when sperm were either centrifuged or not centrifuged, respectively. The substitution of trehalose in the cryopreservation medium for raffinose did not improve recovery of motile cells following cryopreservation (P > 0.05). However, addition of MC resulted in higher percentages of motile sperm after cryopreservation (43 vs 31%; P < 0.05). In addition, sperm viability and acrosomal integrity were simultaneously evaluated using flow cytometry. The addition of both trehalose and MC to media containing acetamide resulted in higher percentages of live acrosome-intact cells than acetamide alone (53 vs 37%; P < 0.05). These results indicate that a combination of permeating and nonpermeating cryoprotectants (acetamide, trehalose and MC) were more effective in preserving rabbit spermatozoa than acetamide alone and that analyzing multiple sperm characteristics, by flow cytometry, can assess sperm damage not detected by analyzing sperm motion characteristics.

Functional analysis using chlortetracycline fluorescence and in vitro fertilization of frozen-thawed ejaculated boar spermatozoa incubated in a protein-free chemically defined medium.

Cumulus-enclosed pig oocytes were matured in vitro, freed from cumulus cells, and inseminated with frozen-thawed ejaculated spermatozoa in a chemically defined protein-free medium containing 37.0 mmol NaHCO3 l-1 and 5 mmol caffeine l-1. When the medium was supplemented with 1 mg polyvinylalcohol (PVA) ml-1, more penetrated oocytes were observed 14 h after insemination with 7-12 x 10(6) cells ml-1 than with 4-5 x 10(6) cells ml-1 and the incidence of polyspermy reflected the sperm concentration used. Varying the NaHCO3 concentration but maintaining the sperm concentration at 8 x 10(6) cells ml-1 resulted in significantly more oocytes being penetrated in media containing 45.83-50.25 than 37.0-41.42 mmol NaHCO3 l-1; there were no significant differences in the incidence of either male pronuclear formation or polyspermy. In medium containing 45.83 mmol NaHCO3 l-1, the inclusion of PVA at 0-5 mg ml-1 had no effect on proportions of penetrated oocytes, male pronuclear formation or polyspermy. However, when spermatozoa from three different boars were evaluated, the penetration and male pronuclear formation rates were highly variable, unlike the incidence of polyspermy. Penetration of cumulus-free oocytes was first detected at 6 h. When spermatozoa were incubated for 6 h in the absence of oocytes, motility, but not vitality, decreased whether or not PVA was included in the medium. Chlortetracycline (CTC) fluorescence analysis of the capacitation state indicated a rapid decline in the proportion of live uncapacitated, acrosome-intact cells and a rapid rise in the proportion of live capacitated, acrosome-reacted cells during the first hour.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca(2+)-regulating mechanisms that modulate bull sperm capacitation and acrosomal exocytosis as determined by chlortetracycline analysis.

We have used chlortetracycline (CTC) analysis to investigate mechanisms that may play important roles during bull sperm capacitation in a culture medium (containing glucose, heparin, and caffeine) known to promote capacitation and fertilization in vitro. In initial experiments employing the Ca2+ ionophore A23187, we identified three discrete CTC patterns so similar to those described for mouse and human sperm that we have employed the same nomenclature: "F," characteristic of uncapacitated, acrosome-intact cells; "B," characteristic of capacitated, acrosome-intact cells; "AR," characteristic of capacitated, acrosome-reacted cells. Over a 60-min period, A23187 stimulated significant increases in B and AR pattern cells, with concomitant decreases in F pattern cells, suggesting a very rapid transition from the uncapacitated to the capacitated state and then on to exocytosis. Without ionophore, significant changes in the proportions of F and B pattern cells were also observed, but the maximum responses required 4 hr; the proportion of AR cells was consistently approximately 15% throughout, indicating a low incidence of spontaneous acrosome loss. Analysis of cells in media with altered composition indicated that the inclusion of either heparin or caffeine significantly promoted capacitation to about the same extent, but together, heparin plus caffeine had an even more stimulatory effect. Despite this, none of these treatments triggered acrosome loss above the levels seen in media lacking these constituents. In the presence of caffeine, with or without heparin, the inclusion of glucose had little effect on responses, but in the presence of heparin there were fewer B cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Andrology: Effects of pentoxifylline and progesterone on human sperm capacitation and acrosome reaction

This study was designed to test the effects of pentoxifylline and progesterone upon capacitation of fresh human spermatozoa. Capacitation and acrosomal integrity were assessed using the fluorescent probe chlortetracycline on spermatozoa co-stained with a supravital fluorescent dye, Hoechst 33258. Hyperactivated motility was measured using computer-assisted movement analysis. After exposure to pentoxifylline (1 mg/ml; 30 min), the fluorescent 'B' pattern, characteristic of capacitated, acrosome-intact cells, increased significantly (P < 0.01), though no increase in 'AR' pattern, characteristic of acrosome-reacted cells, was detected. There was a significant increase in hyperactive motility (P < 0.001). Exposure to progesterone (1 microgram/ml; 60 min) resulted in a significant increase in 'B' pattern (P < 0.05) and 'AR' pattern (P < 0.005), though no effect on the expression of hyperactivation was detected. No effect upon hyperactivation was detected on exposure of fresh or cryopreserved spermatozoa to a physiological range of progesterone concentrations (0.1-1000 ng/ml). Sequential exposure to pentoxifylline then progesterone resulted in a significant increase in 'B' pattern, acrosome loss and hyperactivation. Sperm viability was not affected in any treatment group. These observations suggest that pentoxifylline and progesterone affect capacitation through independent mechanisms. Stimulation of both capacitation and acrosome reaction resulted from sequential exposure to pentoxifylline and progesterone. This may have implications for sperm handling for assisted reproductive techniques.

Ca(2+)-related changes in the capacitation state of human spermatozoa assessed by a chlortetracycline fluorescence assay.

Chlortetracycline (CTC) fluorescence patterns were used to assess Ca(2+)-related changes in the capacitation state of human spermatozoa incubated under conditions that would affect their intracellular Ca2+ levels. Initial experiments were designed to identify consistently occurring patterns and to correlate these with acrosomal status. Incubation for up to 1 h with the ionophore A23187 (10 mumol l-1), known to promote capacitation and acrosomal exocytosis, allowed the identification of three different CTC staining patterns which were very similar to those described for mouse spermatozoa. For this reason, they were given the same nomenclature: 'F'-characteristic of uncapacitated, acrosome-intact cells; 'B'-characteristic of capacitated, acrosome-intact cells; and 'AR'-characteristic of capacitated, acrosome-reacted cells. The distribution of the three patterns in the ionophore-treated suspensions was very different from that in control suspensions treated with dimethylsulfoxide only, with a significantly higher proportion of cells displaying the B and AR patterns and a significantly lower number of cells displaying the F pattern in the ionophore-treated group at all times. A strong concordance was found between the acrosomal status of cells determined using both CTC and fluorescein-conjugated Pisum sativum agglutinin (PSA) staining methods on the same cells. Verification of PSA staining patterns with acrosomal status was obtained by means of transmission electron microscopy. The proportion of cells with uniform fluorescence in the acrosomal region correlated with acrosome-intact cells; those with only equatorial segment staining correlated with fully-reacted cells, and those exhibiting equatorial fluorescence and patchy fluorescence over the rest of the acrosomal region correlated with cells in intermediate stages of exocytosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca(2+)-related changes in the mouse sperm capacitation state: a possible role for Ca(2+)-ATPase.

Mammalian spermatozoa require extracellular Ca2+, some of which must be internalized, to undergo complete capacitation. At a critical threshold, a rise in intracellular Ca2+ will trigger acrosomal exocytosis. We used chlortetracycline (CTC) fluorescence patterns to assess changes in the capacitation state of mouse spermatozoa after incubation under various conditions that would affect their intracellular Ca2+ concentrations. Under standard conditions with 1.80 mmol CaCl2l-1 known to support capacitation within 120 min and subsequent fertilization in vitro, a rise in the number of capacitated, acrosome-intact cells (B pattern) was observed over the first 60 min, followed by a decline. A detectable increase in capacitated, acrosome-reacted cells (AR pattern) coincided with the maximum of B pattern cells and a continued rise was observed over the following 60 min. With incubation in 3.60 mmol Ca2+l-1, the rise in AR cells began at 30 min, suggesting that this treatment accelerates capacitation. Introduction of ionophore A23187 at 15 min to cells in standard Ca2+ produced a similar but even more rapid response, with a maximum in B pattern cells and a noticeable rise in AR cells within 10 min. Thus ionophore-treated cells proceed through capacitation, but do so very quickly. However, ionophore in the presence of 90 mumol Ca2+l-1 could promote transition from the uncapacitated F pattern to the capacitated B pattern, but could not trigger acrosomal exocytosis, indicating that the latter requires high extracellular Ca2+. After preincubation in Ca(2+)-deficient medium, most cells exhibited the uncapacitated F pattern and the introduction of millimolar Ca2+ altered this distribution only slowly, over a period of 50 min. In contrast, preincubation in 90 mumol Ca2+l-1 resulted in a minority of F pattern cells and, within 10 min of millimolar Ca2+ introduction, a significant increase in AR cells was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of capacitation-dependent changes in chlortetracycline fluorescence patterns in mouse spermatozoa requires a suitable glycolysable substrate

Chlortetracycline (CTC) fluorescence patterns were assessed in epididymal mouse sperm suspensions capacitated in exogenous substrate-containing and substrate-free media. A capacitation-dependent transition from a majority of acrosome-intact cells expressing the uncapacitated F pattern of fluorescence to a majority with the capacitated acrosome-intact B and acrosome-reacted AR patterns was confirmed for suspensions incubated a total of 120 min in the presence of a glycolysable substrate, glucose. In contrast, assessment of spermatozoa incubated for 120 min in substrate-free medium revealed a majority of cells with the uncapacitated F pattern, despite an earlier demonstration that such cells are essentially capacitated: upon the introduction of glucose, suspensions are immediately highly fertile. When a suitable glycolysable substrate, either glucose or mannose but not fructose, was added to such suspensions, the distribution of CTC patterns changed within 10 min to a majority of B and AR patterns. Furthermore, the degree of change from uncapacitated to capacitated patterns was substrate concentration-dependent. In contrast, the introduction of the non-metabolizable substrates 2-deoxyglucose and 3-0-methylglucose and the oxidizable substrates sodium pyruvate and sodium lactate caused no change in the patterns from those seen in substrate-free medium. The in-vitro fertilizing ability of sperm suspensions to which increasing amounts of glucose or mannose were added, after initial substrate-free preincubation, directly paralleled the changes in CTC patterns and was as rapid as for suspensions incubated continuously in either hexose. We therefore conclude that the alteration in position of surface components to which CTC binds is not only capacitation-dependent, but also energy-dependent. In the absence of an appropriate exogenous glycolysable substrate, the final transition cannot occur, even though the cells are essentially capacitated.

Development of the acrosome and alignment, elongation and entrenchment of spermatids in procarbazine-treated rats

Intraperitoneally administered procarbazine caused, among other features previously reported (Russell et al., 1983), specific defects in the acrosome of cap phase spermatids of the rat seminiferous epithelium. The effect of procarbazine was to fragment and eventually cause resorption of the acrosomes of a small number of steps 5–9 spermatids. Although the acrosome was lost, dose union of the leaflets of the nuclear envelope underlying the acrosomal sac was maintained as was the marginal fossa and acrosomal zonule. Spermatids at steps 8 and 9 of development, which had lost their acrosomes, showed nuclei which were eccentric within the cell—a feature which normally occurs at these steps of spermiogenesis in acrosome intact cells. Even without an acrosomal sac, the plasma membrane of these cells (in stage VIII) became orientated to the region of the nuclear membrane which would have underlaid the acrosome. Although abundant, Sertoli ectoplasmic specialization did not become aligned with the spermatid head. The spermatid failed to become orientated within the seminiferous epithelium and failed to enter the crypts within the Sertoli cell as usually occurs during the elongation process. Thus, the presence of an acrosome is not likely related to the formation of an eccentric nucleus or the alignment of the surface of the nucleus which would normally underlay the acrosome with the cell's plasma membrane (internal alignment). The presence of an acrosome may be related to the alignment of the spermatid head with the ectoplasmic specialization, which in turn may influence the orientation and positioning of the late spermatids within the seminiferous epithelium (external alignment) and their position within recesses of the Sertoli cell. This study also suggests a role for the manchette in the process of elongation of the spermatid.