Abstract
Cryopreserved spermatozoa demonstrate reduced conception rates compared with fresh spermatozoa when used for artificial insemination. The preliminary stage of cryopreservation of spermatozoa involves cooling to 5 degrees C, during which spermatozoa experience a capacitation-like change, which may be partially responsible for the reduced conception rate observed. The aim of this study was to determine the nature of these capacitation-like changes and how much this process resembles true capacitation. Boar spermatozoa, cooled to 5 degrees C and re-warmed to physiological temperatures (39 degrees C), were compared with spermatozoa capacitated in Tyrode's complete medium (TALP) for 2 h at 39 degrees C. Fluorescent probes, and SDS-PAGE and western blotting were used to visualize events known to occur during capacitation in vitro. Chlortetracycline staining of membrane domains and Fluo-3 detection of changes in intracellular free calcium by flow cytometry in cooled and re-warmed spermatozoa showed similarities to those of capacitated spermatozoa. Alterations to lipid bilayer fluidity assessed by merocyanine fluorescence staining and intracellular signalling pathways detected by tyrosine phosphorylation of cooled and re-warmed spermatozoa, did not completely reflect the changes detected during capacitation in vitro. Thus, cooling spermatozoa to 5 degrees C results in a similar endpoint to that observed in capacitated cells in terms of reactive membranes and changes in intracellular ion concentrations, which may account for their comparable functionality. However, these modifications are not completely analogous and should not be considered true capacitation, but rather a by-passing of the capacitation process.
Highlights
Optimal cryopreservation protocols for mammalian spermatozoa seem able to maintain only approximately 50% survival and it is recognized that the surviving population of spermatozoa is compromised as a result of capacitation-like changes (Watson, 1995)
Spermatozoa incubated in TALP at 39ЊC and assessed at hourly intervals exhibited a progressive increase in the number of B pattern cells and a commensurate decrease in the number of F pattern cells with time (Fig. 1a), whereas there was no change in spermatozoa incubated in PBS
The mechanism that determines the different chlortetracycline staining patterns is unclear; removal or masking of calcium-associated membrane proteins (Caswell and Hutchinson, 1971; Millman et al, 1980; Barboni, 1994) or the complexing of internalized chlortetracycline with Ca2+ (Tsien, 1989) may explain the patterns
Summary
Optimal cryopreservation protocols for mammalian spermatozoa seem able to maintain only approximately 50% survival and it is recognized that the surviving population of spermatozoa is compromised as a result of capacitation-like changes (Watson, 1995) These changes are seen after cooling to 5ЊC (Fuller and Whittingham, 1996, 1997; Cormier et al, 1997). The apparent decrease in conception rates of mammals inseminated with cooled or cryopreserved spermatozoa (Watson, 1979, 1990) can be partially counteracted by inseminating a larger number of spermatozoa (Johnson, 1985), or inseminating closer to the site (boar: Polge et al, 1970; ram: Maxwell et al, 1993) or the time of ovulation (rabbit: Parrish and Foote, 1986; ram: Maxwell et al, 1993; boar: Waberski et al, 1994) These solutions pose problems of practicality and expense.
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