The mammalian spermatozoon

A triple-stain technique has been developed to score normal acrosome-reacted human sperm in fixed smears. Live and dead sperm are first differentiated using the vital stain trypan blue. Sperm are then fixed in glutaraldehyde, dried onto slides, and the postacrosomal region and acrosome are differentiated using Bismark brown and Rose Bengal. Slides are examined at 1,000 X with a bright-field microscope and assessed for 1) the percentage of sperm that were alive at the time of fixation and 2) the percentage of sperm that had undergone normal acrosome reactions. Experiments are included that show that trypan blue is a reliable stain for dead sperm and that Rose Bengal stains only sperm having intact acrosomes. This technique may have applications in experimental and clinical studies on sperm capacitation, acrosome reactions, and fertilization in laboratory and domestic animals as well as in man.

A triple-stain technique for evaluating normal acrosome reactions of human sperm.

Summary
The mammalian sperm acrosome reaction is a unique form of exocytosis, which includes the loss of the involved membranes. Other laboratories have suggested the involvement of hydrolytic enzymes in somatic cell exocytosis and membrane fusion, and in the invertebrate sperm acrosome reaction, but there is no general agreement on such an involvement. Although reference was made to such work in this review, the focus of the review was on the evidence (summarized below) that supports or fails to support the importance of certain hydrolytic enzymes to the mammalian sperm acrosome reaction. Because the events of capacitation, the prerequisite for the mammalian acrosome reaction, and of the acrosome reaction itself are not fully understood or identified, it is not yet always possible to determine whether the role of a particular enzyme is in a very late step of capacitation or part of the acrosome reaction.

(1) The results of studies utilizing inhibitors of trypsin-like enzymes suggest that such an enzyme has a role in the membrane events of the golden hamster sperm acrosome reaction. The enzyme involved may be acrosin, but it is possible that some as yet unidentified trypsin-like enzyme on the sperm surface may play a role in addition to or instead of acrosin. Results obtained by others with guinea pig, ram and mouse spermatozoa suggest that a trypsin-like enzyme is not involved in the membrane events of the acrosome reaction, but only in the loss of acrosomal matrix. Such results, which conflict with those of the hamster study, may have been due to species differences or the presence of fusion-promoting phospholipase-A or lipids contaminating the incubation media components, and in one case to the possibly damaging effects of the high level of calcium ionophore used. The role of the trypsin-like enzyme in the membrane events of the hamster sperm acrosome reaction may be to activate a putative prophospholipase and/or to hydrolyse an outer acrosomal or plasma membrane protein, thus promoting fusion. A possible role of the enzyme in the vesiculation step rather than the fusion step of the acrosome reaction cannot be ruled out at present.

(2) Experiments utilizing inhibitors of phospholipase-A2, as well as the fusogenic lysophospholipid and cis-unsaturated fatty acid hydrolysis products that would result from such enzyme activity, suggests that a sperm phospholipase-A2 is involved in the golden hamster sperm acrosome reaction. Inhibitor and LPC addition studies in guinea pig spermatozoa have led others to the same conclusion. The fact that partially purified serum albumin is important in so many capacitation media may be explained by its contamination with phospholipase-A and/or phospholipids. Serum albumin may also play a role, at least in part, by its removal of inhibitory products released by the action of phospholipase-A2 in the membrane. The demonstration of phospholipase-A2 activity associated with the acrosome reaction vesicles and/or the soluble component of the acrosome of hamster spermatozoa, and the fact that exogenous phospholipase A2 can stimulate acrosome reactions in hamster and guinea pig spermatozoa, also support a role for the sperm enzyme. The actual site or the sites of the enzyme in the sperm head are not yet known. The enzyme may be on the plasma membrane as well as, or instead of, in the acrosomal membranes or matrix. A substrate for the phospholipase may be phosphatidylcholine produced by phospholipid methylation. It is possible that more than one type of ‘fusogen’ is released by phospholipase activity (LPC and/or cis-unsaturated fatty acids, which have different roles in membrane fusion and/or vesiculation.

In addition to acting as a potential ‘fusogen’, arachidonic acid released by sperm phospholipase-A2 probably serves as precursor for cyclo-oxygenase or lipoxygenase pathway metabolites, such as prostaglandins and HETES, which might also play a role in the acrosome reaction. Although much evidence points to a role for phospholipase-A2, phospholipase-C found in spermatozoa could also have a role in the acrosome reaction, perhaps by stimulating events leading to calcium gating, as suggested for this enzyme in somatic secretory cells.

(3) A Mg2+-ATPase H+-pump is present in the acrosome of the golden hamster spermatozoon. Inhibition of this pump by certain inhibitors of ATPases (but not by those that only inhibit mitochondrial function) leads to an acrosome reaction only in capacitated spermatozoa and only in the presence of external K+. The enzyme is also inhibited by low levels of calcium, and such inhibition, combined with increased outer membrane permeability to H+ and K+, and possibly plasma membrane permeability to H+ (perhaps by the formation of channels), may be part of capacitation and/or the acrosome reaction. The pH of the hamster sperm acrosome has been shown to become more alkaline during capacitation, and such a change may result in the activation of hydrolytic enzymes in the acrosome or perhaps in a change in membrane permeability to Ca2+. A similar Mg2+-ATPase has not been found in isolated boar sperm head membranes. However, that conflicting result could have been due to the use of noncapacitated boar spermatozoa for the preparation of the membranes or to protease modification of the boar sperm enzyme during assay.

(4) Inhibition of Na+, K+-ATPase inhibits the acrosome reaction of golden hamster spermatozoa, and the activity of this enzyme increases relatively early during capacitation. A late influx of K+ is important for the acrosome reaction. However, this late influx may not be due to Na+, K+-ATPase, but instead may be due to a K+ permeability increase (possibly via newly formed channels) in the membranes during capacitation. It is suggested in this review that Na+, K+-ATPase has a role early in capacitation rather than directly in the acrosome reaction (although such a role cannot yet be completely ruled out). One possible role for the enzyme in capacitation might be to stimulate glycolysis (which appears to be essential for capacitation and/or the acrosome reaction of hamster and mouse spermatozoa). The function of the influx of K+ just before the acrosome reaction is probably to stimulate, directly or indirectly, the H+-efflux required for the increase in intraacrosomal pH occurring during capacitation. Direct stimulation of the acrosome reaction by a change in membrane potential resulting directly from K+-influx is not a likely explanation for the hamster results. However, the importance of an earlier membrane potential change, due to increased Na+, K+-ATPase during capacitation, and/or of later membrane potential changes resulting from the pH change, cannot be ruled out. Although K+ is required for the hamster acrosome reaction, other workers have reported that K+ inhibits guinea pig sperm capacitation. However, the experimental procedures used in the guinea pig sperm studies raise some questions about the interpretation of those inhibition results.

(5) Ca2+-influx is known to be required for the acrosome reaction. Others have suggested that increased Ca2+-influx due to inhibition or stimulation of sperm membrane calcium transport ATPases are involved in the acrosome reaction. There is as yet no direct or indirect biochemical evidence that inhibition or stimulation of such enzymatic activity is involved in the acrosome reaction, and further studies are needed on those questions.

(6) I suggest that the hydrolytic enzymes important to the hamster sperm acrosome reaction will also prove important for the acrosome reaction of all other eutherian mammals.

The importance of hydrolytic enzymes to an exocytotic event, the mammalian sperm acrosome reaction

Significance of the equatorial segment of the acrosome of the spermatozoon in eutherian mammals

Human spermatozoa display unusually limited affinities in their interaction with oocytes of other species. They adhered to and, when capacitated, penetrated the vestments of the oocyte of an ape--the gibbon, Hylobates lar--both in vivo and in vitro. On the other hand, human spermatozoa would not even attach to the zona surface of sub-hominoid primate (baboon, rhesus monkey, squirrel monkey), nor to the non-primate eutherian oocytes tested. Among the apes the gibbon stands furthest from man. Thus, although the specificity of human spermatozoa is not confined to man alone, it probably is restricted to the Hominoidea. This study also suggests that the evolution of man and perhaps the other hominids has been accompanied by a restrictive change in the nature of the sperm surface which has limited and made more specific the complementary surface to which their spermatozoa may adhere. For the failure of human spermatozoa to attach to the zona surface of all non-hominoid oocytes stands in contrast to the behaviour of spermatozoa of the several other mammals studied which, in most combinations, adhered readily to foreign oocytes, including those of man. Taxonomically, the demonstration of a compatibility between the gametes of man and gibbon, not shared with cercopithecids, constitutes further evidence for inclusion of the Hylobatidae within the Hominoidea.

C. Barros

Papers

The Acrosomal Region and the Acrosome Reaction in Sperm of the Golden Hamster