ASSESSMENT OF THE ACROSOMAL STATUS OF MEMBRANE-INTACT RAM SPERMATOZOAAFTER FREEZING AND THAWING, BY SIMULTANEOUS LECTIN HOECHST-33258 STAINING

We have evaluated the effect of freezing and thawing on the acrosomal status of ram spermatozoa, especially those that withstood cryopreserv ation as assessed by membrane integrity. To this end, we performed sim ultaneous lectin/Hoechst 33258 staining, and compared the ability of t hree fluoresceinated lectins. Ram spermatozoa were treated with fluore scein isothiocyanate-labelled Pisum sativum lectin (PSA), fluorescein isothiocyanate-labelled Arachis hypogea lectin (PNA) and fluorescein i sothiocyanate-labelled Triticum vulgaris lectin (WGA) and simultaneous ly with Hoechst 33258 for determination of membrane integrity and acro somal status. In all cases, three forms were readily distinguished by their distribution pattern. For both PSA and PNA, the most abundant fo rm found in fresh semen consisted of fluorescence on the acrosomal are a. This form corresponds to acrosome-intact spermatozoa, as assessed b y Differential Interference. Contrast (DIG) microscopy. Two minor form s showed weak fluorescence on the equatorial segment or no fluorescenc e on the head. DIC microscopy revealed that both forms were associated with acrosome-lost spermatozoa. WGA labelling showed two forms, one o f which consisted of fluorescence on the entire head, albeit more inte nsely on its anterior segment. Spermatozoa in this form were acrosome- intact by DIG. The other form lacked fluorescence on the acrosomal reg ion, but still showed faint fluorescence in the posterior region. This form was acrosome-lost by DIG. Incubation of fresh spermatozoa with c alcium ionophore A23187 for up to 1 h significantly increased the perc entage of those forms identified as acrosome-reacted as described abov e. This was confirmed by the time-dependent accumulation of these form s, as well as by DIC microscopy. At all times, differences among value s obtained using these three lectins were not significant. Freezing an d thawing led to a decrease of both membrane integrity and acrosomal i ntegrity, irrespective of the lectin used. However, almost all spermat ozoa that withstood cryopreservation, as evaluated by Hoechst exclusio n, showed intact acrosomes. In this case, no differences between fresh and frozen/thawed samples were observed. These results suggest that t he structural integrity of ram spermatozoa is mostly unaffected after cryopreservation, suggesting that it is damage to the plasma membrane that is primarily responsible for the low fertility of cryopreserved s amples.