however, are not determined stochastically. The fact that indiÕiduals can often be classified as ‘‘good freezers’’ or ‘‘bad freezers’’ implies that certain characteristics of
membrane structure, which may be genetically determined, predispose towards survival under cryopreservation stress. Attempts to modify lipid composition have not demon-
strated any dramatic benefits, perhaps implying that other membrane elements may be more important, e.g. cytoskeleton. This is an area for further investigation.
Nevertheless, even if we optimise the process and minimise the cell death, there will still be a proportion of cells which fail to survive. We need, therefore, to concentrate on
the function of the surviving population.
3. Increasing the quality of survivors
3.1. Capacitation-like changes A significant breakthrough in understanding has come with the recognition that
Ž .
cooled and rewarmed spermatozoa behave as if they were capacitated Watson, 1995 . However, we are unsure at present whether spermatozoa manifest the changes of
capacitation or simply are enabled to by-pass capacitation and proceed directly to acrosomal exocytosis. We have begun to study this phenomenon and have shown that
cooled spermatozoa display chlortetracycline staining and an increase in intracellular free Ca
2q
, typical of capacitated spermatozoa, but that the tyrosine phophorylation Ž
patterns are somewhat different in cooled and rewarmed spermatozoa Watson and .
2q
Green, in press . Fig. 2 shows the free Ca in cooled spermatozoa compared with that
seen in capacitated spermatozoa. We are continuing to investigate these events to determine the nature of the membrane changes resembling capacitation.
Fig. 2. The intracellular free Ca
q
response of boar spermatozoa either to incubation in a capacitating medium or to cooling. The illustrations are of ‘‘dot plots’’ from the flow cytometer of spermatozoa labelled with the
Ž .
viability probe, propidium iodide vertical axis, FL3 LOG and a calcium-sensitive indicator, Fluo-3 AM ester Ž
. Ž . Ž .
horizontal axis, FL1 LOG . A Control at time zero, B after 2-h incubation at 39 8C in modified Tyrode’s
Ž .
Ž . medium incubation at ambient temperature does not produce this change , and C after slow cooling at
0.18 8Crmin to 58C and rapid rewarming to 398C. Area P represents dead cells, area Q represents cells with a
low calcium concentration and area R represents those with high calcium concentration. A strong similarity Ž .
Ž . can be seen in the responses in B and C , indicating a rise in intracellular free calcium in both situations in
the majority of live cells.
3.2. Motility impairment One obvious characteristic of cryopreserved spermatozoa is the decline in motility of
the cells. While a minority seem to exhibit vigorous forward progression, the majority show a variable degree of impairment. This would seem to be an important contribution
to their relatively poor fertilising potential when introduced into the reproductive tract at artificial insemination. A study of cryopreserved human spermatozoa under IVF condi-
Ž tions found that both motility and forward progression were important factors Kelly et
. al., 1997 . However, one recent observation reached a different conclusion; in a study of
in vitro fertility of asthenoteratozoospermic dogs, the quality of motility though poor did Ž
. not impair fertility Hewitt and England, unpublished . These studies relate only to
events associated with the final contact with the oocyte; transport to the site of fertilisation may still require a minimum capability of forward motility, which may be
compromised in the majority of cryopreserved spermatozoa.
3.3. OxidatiÕe damage More important is the observation that oxidative damage could impair sperm func-
tion. This subject is difficult to tackle because of the recognition that a degree of Ž
superoxide radical formation is necessary for the events preceding fertilisation de .
Lamirande et al., 1997 . However, it is now generally accepted that cryopreservation induces the formation of reactive oxygen species that are detrimental to subsequent
Ž .
performance Alvarez and Storey, 1992; Bell et al., 1993; O’Flaherty et al., 1997 . Much of the published work on lipid peroxidation and cryopreservation relates to work on
human spermatozoa. The use of antioxidants in cryopreservation diluents is not commonplace, partly
because there are a number of possible agents from which to select, and partly because Ž
. different pathways and hence, different antioxidants are utilised in different species
Ž .
Askari et al., 1994; O’Flaherty et al., 1997 . Moreover, it is clear that oxidative damage Ž
is only one of a number of stresses the cryopreserved sperm cell experiences Alvarez .
and Storey, 1993 . This is clearly an area for further investigation. 3.4. Surface changes affecting recognition of receptors
An issue that has been recently examined is the extent to which the surviving Ž
population is impaired in its ability to interact with the oviductal epithelium Ellington et .
al., 1999 . These interactions are now recognised as receptor–ligand interactions, often involving intracellular signalling mechanisms, which are actively being explored. A
Ž similar interaction involves spermatozoa and the oocyte and its vestments Bwanga et
. al., 1991; Oehninger et al., 1993 . The freeze-fracture observations, suggesting that the
clustering of membrane proteins during lipid phase separations induced by cooling are Ž
. not entirely reversible see above , may well have implications for receptor–ligand
interactions. Although the evidence from IVF studies generally indicates that cryopre- Ž
. served spermatozoa are capable of effecting fertilisation e.g., Donaghue et al., 1992 ,
this is an artificial situation when hundreds of spermatozoa are present. In vivo, the
reality may be quite different when only a few spermatozoa reach the site of fertilisation Ž
. see above .
3.5. Ability of sperm to sustain embryonic deÕelopment Finally, another area that has not been adequately studied is the ability of cryopre-
served spermatozoa to sustain embryonic development. Technically, this is difficult to investigate since the evidence of early embryonic death often passes without overt signs.
Nevertheless, a persistent suggestion is that frozen–thawed spermatozoa are associated Ž
. with an increased incidence of early embryonic mortality Salamon and Maxwell, 1995 .
The potential mechanisms can now be studied more effectively. DNA damage can be Ž
. investigated with the COMET assay Hughes et al., 1996 or with flow cytometry
Ž .
Karabinus et al., 1990; Royere et al., 1991 and may indicate functional damage to the Ž
. nuclear structures Ellington et al., 1998 . Moreover, the suggestion that the spermato-
Ž zoon’s contribution to the zygote is more than the haploid male genome Navara et al.,
. 1995 implies that there are more subtle ways in which the spermatozoon’s function
could be disrupted during cooling and cryopreservation. The post-syngamy fate of other Ž
sperm structures is now known better than ever before Sutovsky et al., 1996; Sathanan- .
Ž than et al., 1997 , and the possible importance of sperm RNA Rohwedder et al., 1996;
. Miller, 1997
to the events before the embryonic genome is activated cannot be disregarded. Any of these structures may suffer alteration during cryopreservation and
can all now be investigated.
4. Conclusion
