Assessment of frozen–thawed semen

6. Assessment of frozen–thawed semen

While it is obvious that freezing and thawing causes sperm damage, it is less apparent that some relatively minor damaging effects may entirely abolish the fertility of individual spermatozoa. In order to fertilize an egg, a spermatozoon must retain the capacity to reach and penetrate the oocyte, and thus needs not only a functional flagellum for propulsion but also the appropriate membrane surface chemistry to avoid either engulfement by phagocytes in the female reproductive tract or irreversible binding by epithelial cells. Furthermore, the spermatozoon must be capable of binding to the zona pellucida and responding with an acrosome reaction when the appropriate sig- nalling pathway is stimulated. This implies in turn that the signalling pathway itself, and the associated structures such as the plasma and outer acrosomal membranes, must remain intact and undamaged throughout cryopreservation. Zona penetration is followed by sperm–oolemma fusion. The mechanism for egg activation, which either involves Ž . transfer of a cytoplasmic factor oscillin; Parrington et al., 1996 or stimulation of another signalling pathway, must also remain undamaged. Finally, the sperm chromatin should be available for decondensation, not having suffered inappropriate additional stabilization or destabilization during cryopreservation. Damage to any one of the physiological units that govern these attributes would render a spermatozoon unable to fertilize the oocyte; the spermatozoon would be infertile. This level of subtlety in determining the proportion of spermatozoa which remain completely unaffected by cryopreservation would explain the surprising discrepancy in sperm numbers needed to achieve comparable fertility rates with fresh or frozen bull sperm; 10 times more frozen Ž . than fresh spermatozoa were required Shannon, 1978 . At the time that data were published, standard sperm doses per insemination were 25 million frozen and 2.5 million fresh spermatozoa. Although greater economy of sperm dose has been developed in the intervening years, the same 10:1 ratio still holds; sperm doses in New Zealand are now 10–15 million cryopreserved spermatozoa per insemination straw vs. 1–1.5 million Ž . fresh spermatozoa Vishwanath et al., 1996 . 6.1. Sperm function tests Many technical approaches to sperm function testing have been developed over the Ž past 10–15 years for reviews, see Amann, 1989; Critser and Noiles, 1993; Oehninger et . al., 1992 , and it is increasingly possible to combine the techniques in order to assess different aspects of function simultaneously. The functional aspects most readily exam- ined include plasma membrane, acrosomal and mitochondrial integrity, ability to undergo the acrosome reaction in response to appropriate stimuli and sperm motility. Flow cytometry is frequently used as an alternative to microscopy, but the latter is still essential if details of cell structure have to be examined. Although the eosinrnigrosin technique is still used in many routine laboratories as a means of plasma membrane assessment, it is being superseded by fluorescent tests Ž . Harrison and Vickers, 1990; Johnson et al., 1996 . Typically, these involve the Ž . exclusion of a membrane-impermeant fluorescent dye, such as propidium iodide PI , by intact cells and the simultaneous uptake and retention of a different fluorochrome. The latter can either be the non-fluorescent ester of a fluorochrome, e.g. carboxyfluorescein diacetate, which generates the fluorochrome upon intracellular cleavage by an esterase, or a membrane-permeant fluorescent compound which has affinity for DNA, such as Ž . SYBR-14 Garner and Johnson, 1995 . Damaged and intact cells are therefore revealed in different colours, and the proportion of intactrdamaged cells, sometimes called the liverdead ratio, is estimated by either microscopy or flow cytometry. Mitochondrial assessment can be estimated using rhodamine 123, a fluorescent dye that binds to the inner mitochondrial membrane. The mechanisms involved in some of the fluorescent methods, and consequently their validity, have sometimes been questioned. For instance, when stained by the SYBR- 14rPI technique, the intact cell nuclei appear green, since SYBR-14 binds to chromatin. Cells change colour and nuclei become red once membrane damage has occurred. Ž . Johnson et al., 1996 discussed these changes in terms of ‘‘DNA viability’’ and membrane potential, but in this instance the mode of action remains unclear. This particular method has nevertheless proved useful in conjunction with freeze–thaw techniques as cell damage can be detected directly even in the presence of egg yolk Ž . Medrano and Holt, 1996 . Some techniques seem difficult to implement in a given laboratory. The fluorescent dye Hoechst 33258, which is unable to permeate the plasma membrane of intact cells, has been used as a marker of cell membrane integrity in Ž . glutaraldehyde-fixed cells De Leeuw et al., 1991 . This technique has proved somewhat idiosyncratic in the hands of different groups, even though it has the potential advantage of being combined with other fluorescent probes for the determination of acrosomal status. Rhodamine 123 is regarded as binding only to actively metabolizing mitochon- Ž dria; however, since sperm mitochondria continue to fluoresce below y208C Holt et . al., 1988 when viewed directly by cryomicroscopy, it seems likely that it will continue to fluoresce once the dye is bound even if metabolic activity is extremely low. Since cryopreservation induces physical damage in some cells, the proportion of undamaged acrosomes is widely assessed. As acrosomal integrity does not necessarily reflect plasma membrane integrity, combining the two tests provides a more discrimina- tory assessment than either test on its own. Although phase contrast microscopy of fixed spermatozoa has been used extensively to evaluate spermatozoa, generating data about Ž . the proportions of normal acrosomal ridges NAR seen in many publications, this approach has been largely overtaken by the use of fluorescent probes. Many lectins, proteins that interact with the glycoconjugates of the acrosomal membranes or matrix, Ž . have been tested for use in acrosomal evaluation for review, see Cross, 1995 . These include peanut agglutinin, Concanavalin A, Pisum sativum agglutinin and Ricinus communis agglutinin. Monoclonal antibodies are also used for acrosomal evaluation, but they are more time consuming. Using these techniques with cells cryopreserved in egg yolk diluents requires a post-thaw washing step, otherwise, the yolk interferes with lectin or antibody binding. This practical issue probably means that the frequency of damaged acrosomes is always artificially increased by the washing procedure. Acrosomal and plasma membrane integrity can be regarded functionally as well as in terms of structure. The combined ability of the acrosome-plasma membrane complex to respond to acrosome reaction inducers, or to bind and penetrate the zona pellucida is an Ž . important attribute of cell function. Whitfield and Parkinson 1992 used heparin to induce the acrosome reaction in bull spermatozoa. They observed a high correlation between 90-day non-return rates and the extent of acrosome reaction induction in a fertility trial using frozen semen. This study was remarkable in that the correlation was found even though the acrosome reactions were detected with unfrozen spermatozoa, suggesting that the freezing itself did not change the responsiveness of the structurally intact cells, and implying that acrosome reacting ability may be correlated with the capacity to reach the site of fertilization. A similar approach was taken by Graham and Ž . Foote 1987 , who used liposomes instead of heparin to induce acrosome reactions. In human clinical investigations, the principle of examining acrosomal responses to various inducing substances, e.g. ionophore A23187 and more recently progesterone, has been developed although its validity has yet to be confirmed. This type of approach has been combined with the zona-free hamster egg penetration test to assess acrosomal respon- siveness and ability to fuse with the oolemma. Ž . An indirect test of capacitation status, using the antibiotic chlortetracycline CTC , Ž has recently provided a new insight into the functional status of spermatozoa for . review, see Fraser, 1995 . With CTC as a fluorescent probe, spermatozoa show various fluorescent patterns which have been correlated with predominantly incapacitated, capacitated or acrosome reacted sperm populations. Cooled and frozen–thawed sperma- Ž tozoa apparently become more ‘‘capacitated’’ as determined by this test; Watson 1995, . 1996 has argued that this effect shows that the cryopreservation process is in some respects analogous to capacitation, which may account for the shortened lifespan of spermatozoa, and the brief period of capacitation required to promote fertilization in vitro, after freeze-thawing. A slightly different approach to testing the functional integrity of the plasma membrane-acrosome complex has been evaluation of the sperm interaction with isolated zonae pellucidae. As acrosome reactions are physiologically induced by zona pellucida proteins, incubation of spermatozoa with fresh, frozen or salt-stored zonae has been developed as a test. The end-points can be either the number of sperm bound to the zona surface, or the number which undergo the acrosome reaction and commence penetration Ž . see, for example, Fazeli et al., 1993 . This type of assay was developed further with the Ž . introduction of the hemi-zona assay Fazeli et al., 1995 , where one-half of a zona pellucida is reacted with a reference semen sample while the other is reacted with the sample under test. As acrosomes contain proacrosin, the precursor of acrosin which is implicated in zona binding and penetration, it has been suggested that spontaneous conversion of proacrosin to acrosin during cryopreservation may depress fertility. A recent study Ž . Palencia et al., 1996 , using a labelled serine protease-inhibitor to detect active acrosin, failed to confirm this hypothesis but introduced a potentially important novel approach to acrosomal assessment which is amenable to flow cytometry as well as microscopy. Evaluation of post-thaw sperm motility is widely used for post-thaw semen assess- ment, and in its simplest form provides a rough guide to the survival of spermatozoa. However, in its more sophisticated applications, for example using computerized semen analysis systems, the interpretation of derived measurements is highly dependent on the existence of data from fertility trials. For human spermatozoa, the significance of linear progression was shown to be significantly predictive of the fertilizing capacity of donor Ž . semen samples in a fertility clinic Holt et al., 1985, 1989; Irvine and Aitken, 1986 . Amplitude of lateral head displacement, an indicator of the power exerted by the flagellum, has also been identified as being predictive of fertility, and the ability of Ž . individual spermatozoa to penetrate cervical mucus Jeulin et al., 1986 . It seems likely that this type of analysis is still at an early stage of development, and that considerably more information will become available when methods for the analysis of sperm population data are finalized. Most studies have worked with population mean values, which may well obscure the true subpopulation structures to be found within samples. Recently, efforts to develop multivariate clustering techniques for the analysis of sperm Ž . Ž . populations have made some progress see, Holt, 1996 . Davis et al. 1995 used pre-freeze kinematic measurements as predictors of the proportion of spermatozoa that would survive cryopreservation. Few similar studies have been applied to animal Ž . spermatozoa and the results are conflicting. Bailey et al. 1994 were unable to show that computer-assisted semen analysis could predict the fertility of frozen bull semen, but recent studies with liquid-stored boar semen demonstrated that measurements of sperm survival in vitro under capacitating conditions, were indicative of conception rate Ž . and litter size Holt, 1995 . The above description of sperm function tests is not intended to be exhaustive. However, it would not be complete without mentioning that the relative values of some Ž of these functional tests have been examined in heterospermic insemination trials for . review, see Dziuk, 1996 . This approach involves inseminating doses of semen that originate from different males, then comparing relative fertility values with the outcomes of the laboratory tests. Using this technique to study cryopreserved boar semen, Ž . Hammitt et al. 1989 obtained high correlations between fertility and the zona-free hamster egg penetration test, sperm motility in the presence and absence of caffeine, and the proteolytic activity of individual acrosomes. Interestingly, acrosomal morphology Ž . showed no correlation with relative fertility. When Budworth et al. 1988 studied the motility of frozen-thawed bull semen, no significant relationships between objectively measured sperm motion parameters and the results of homospermic inseminations were Ž . detected all r values - 0.1 . However, the motion parameters:curvilinear velocity Ž . Ž . VCL and straightline velocity VSL accounted for approximately 70 of variance in competitive fertility index under the heterospermic insemination protocol. Such contrast- ing relationships must point to fundamental differences between the biology of the two systems, and suggest that the two types of test are addressing different questions. With homospermic inseminations, the question is ‘‘will conception occur, and if so, how many eggs will be fertilized?’’, while for heterospermic inseminations, there is an implicit assumption that conception will definitely occur, and the question is then ‘‘which subpopulation of sperm will be more successful?’’. Success in the competitive situation may therefore be determined by factors that control speed of capacitation, rate of sperm transport and precedence in colonization of the oviduct. It follows that spermatozoa that fare poorly in a competitive sense may nevertheless be fertile in the absence of competing spermatozoa; but absolute fertility may then depend more upon their longevity in the female tract. Continued analyses of these issues, using various types of fertility trials, may help with the development of more exacting sperm function tests. It is dangerous to conclude that any single current test can predict fertility with much accuracy; however, it is probably reasonable to remark that the use of such tests in combination can help to identify subfertile individuals, ejaculates or cryopreserved samples, and can therefore be regarded as useful for quality assurance.

7. Concluding remarks