The spermatozoa of most invertebrates (e.g., sea urchins) and nonmammalian vertebrates (e.g., fishes and amphibians) have full capacity to fertilize eggs upon leaving the testis. Testicular spermatozoa of mammals, on the other hand, do not possess the ability to do so. Their fertilizing capacity develops as they pass through the epididymis (Young, 1931; Nishikawa and Waide, 1952; Blandau and Rumery, 1964; Bedford, 1966; Orgebin-Crist, 1967). This process, apparently “unique” to mammals, is referred to as the epididymal maturation of spermatozoa. Even after their maturation, however, spermatozoa require an additional phase of maturation or capacitation within the female genital tract before they are able to fertilize eggs (Austin, 1951, 1952; Chang, 1951a). Thus, epididymal maturation and capacitation are two extra steps that mammaliam spermatozoa must take before they can effect fertilization. In this chapter, I will discuss how mammalian spermatozoa prepare themselves for fertilization and how the spermatozoa and eggs interact during fertilization. The process and mechanisms of sperm transport in the female genital tract will not be dealt with extensively here. Readers are referred to Bishop (1961, 1969), Blandau (1969), Bedford (1970b, 1972b), Thibault (1972, 1973a), Zamboni (1972), Blandau and Gaddum-Rosse (1974), Hafez and Thibault (1975), Overstreet and Katz (1977), Overstreet and Cooper (1978, 1979b), Overstreet et al. (1978), Shalgi and Kraicer(1978), Cooper et al. (1979) and Hunter (1975, 1980). The rejection or elimination of extra spermatozoa by the fertilized egg, one of the most fascinating events in fertilization, will not be discussed here. Instead, readers are referred to the chapter by Dr. Wolf in this volume. The physiology of egg activation has been described and discussed to some extent by Gwatkin (1977) and Yanagimachi (1978a). There are numerous reviews dealing with general aspects of mammalian fertilization. The following are recommended to aid in grasping the outline of mammalian fertilization: Austin and Bishop (1957), Austin and Walton (1960), Austin (1961, 1968), Blandau (1961), Piko (1969), Thibault (1969), Bedford (1970a,b, 1972b), Gwatkin (1976, 1977), Yanagimachi (1977, 1978a), Bedford and Cooper (1978) and Hunter (1980).

Mechanisms of Fertilization in Mammals

We describe two methods for detecting acrosome reactions of human sperm at the light microscopic level. The techniques include the use of a supravital stain to detect dead sperm in order to differentiate between “physiological” and “degenerative” acrosome reactions. Sperm are incubated with the supravital stain Hoechst 33258 (a fluorescent DNA-binding dye with limited membrane permeability), washed, suspended in 95% ethanol for fixation and permeabilization, and dried onto slides. The sperm are then labeled either by indirect immunofluorescence using rabbit anti-human sperm antiserum or with fluoresceinated Pisum sativum agglutinin (PSA). Both probes intensely label the acrosomal region of acrosome-intact sperm. Electron microscopy revealed the major site of PSA binding to be the acrosomal contents. Acrosome-reacted sperm have diminished acrosomal labeling by both probes; sperm with nuclei labeled by Hoechst stain are considered nonviable, and are excluded from the assay. Both assays are rapid, give similar results, and detect an increase in acrosome reactions following exposure to the ionophore A23187.

Two simple methods for detecting acrosome‐reacted human sperm

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.

The abundance of data pertaining to the metabolism of lipids in relation to mammalian fertilization has warranted an effort to assemble a molecular membrane model for the comprehensive visualization of the biochemical events involved in sperm capacitation and the acrosome reaction. Derived both from earlier models as well as from current concepts, our membrane model depicts a lipid bilayer assembly of space-filling molecular models of sterols and phospholipids in dynamic equilibrium with peripheral and integral membrane proteins. A novel feature is the possibility of visualizing individual lipid molecules such as phosphatidylcholine, phosphatidylethanolamine, lysophospholipids, fatty acids, and free or esterified cholesterol. The model illustrates enzymatic reactions which are believed to regulate the permeability and integrity of the plasma membrane overlying the acrosome during interactions between the male gamete and capacitation factors present in fluids of the female genital tract. The use of radioactive lipids as molecular probes for monitoring the metabolism of cholesterol and phosphatidylcholine revealed the presence of (1) steroid sulfatase in hamster cumulus cells, (2) lecithin: cholesterol acyltransferase in human follicular fluid, (3) phospholipase A2, and (4) lysophospholipase in human spermatozoa. These enzymatic reactions can be integrated into a pathway that provides a link between the concepts of lysophospholipid accumulation in the sperm membranes and alteration of the cholesterol/phospholipid ratio as factors involved in the preparation of the membranes for the acrosome reaction. Capacitation is viewed as a reversible phenomenon which, upon completion, results in a decrease in negative surface charge, an efflux of membrane cholesterol, and an influx of calcium between the plasma and outer acrosomal membranes. Triggered by the entry of calcium, the acrosome reaction involves phospholipase A2 activation followed by a transient accumulation of unsaturated fatty acids and lysophospholipids implicated in membrane fusion which occurs during the formation of membrane vesicles in spermatozoa undergoing the acrosome reaction.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/mrd.1120120209

A molecular membrane model of sperm capacitation and the acrosome reaction of mammalian spermatozoa

In order to fertilize, the spermatozoa of eutherian mammals must undergo capacitation in the female tract. The subcellular changes involved in capacitation finally seem to permit the influx of Ca2+ required for onset of the acrosome reaction, and they result also in a hyperactivated form of motility. However, why capacitation has appeared as an essential prerequisite for eutherian fertilization is unknown. Both these facets of capacitation may reflect new cellular control mechanisms for regulating the sperm's activities, necessitated by evolutionary change in the oocyte. The first may reflect a loss of the oocyte's stimulation of the acrosome reaction, and the second a concomitant appearance of unusually formidable egg vestments the spermatozoon must penetrate. Coordination of the rate of capacitation appropriate to fertilization in vivo may depend not only on the minimal time ordained for the species, but also on a heterogeneity among subpopulations of spermatozoa within any one sample and the timing of sperm transport to the oviduct.

Significance of the need for sperm capacitation before fertilization in eutherian mammals.

The role of Ca+2 in the acrosome reaction of echinoid and mammalian sperm was investigated using the Ca+2 transporting ionophore A23187. The ionophore induced morphologically normal acrosome reactions in both types of sperm (as assessed by electron microscopic observation of echinoid sperm and phase contrast microscopic observation of mammalian sperm). In echinoids, these reactions were immediate. In the guinea pig and hamster, ionophore significantly decreased the capacitation interval; early reactions were accompanied by activation of motility. Ionophore induced reactions were affected by sperm, ionophore and Ca+2 concentrations. Since both ionophore induced and natural reactions require extracellular Ca+2, it is suggested that an influx of Ca+2 represents the initial step of the acrosome reaction. Under natural conditions, the permeability change which results in Ca+2 influx may be induced in echinoid sperm by egg jelly and may occur in mammalian sperm during capacitation. Ionophore A23187 should prove an experimentally useful drug for further study of the acrosome reaction since its effect on cells is understood, it induces synchronous reactions in a high percentage of sperm, and it conveniently reduces the capacitation interval in mammalian sperm.

The role of calcium in the acrosome reaction: An analysis using ionophore A23187

Ionophore A23187, in the presence of extracellular Ca+2, induces a morphologically normal acrosome reaction in sperm of the sea urchin and precocious acrosome reaction and activation of guinea pig sperm. Increased membrane permeability to Ca+2 is responsible for initiating the acrosome reaction in both sea urchin and guinea pig sperm. In sea urchin sperm, the permeability change is brought about by egg jelly, whereas in the guinea pig sperm it accompanies capacitation.

B. L. Hylander

Papers

The role of calcium in the acrosome reaction: An analysis using ionophore A23187

Ionophore A23187 induces acrosome reactions in sea urchin and guinea pig spermatozoa.