Physiology and pathophysiology of the human spermatozoon: the role of electron microscopy.

JOURNAL OF ELECTRON MICROSCOPY TECHNIQUE 17:412-436 (1991)

Physiology and Pathophysiology of the Human Spermatozoon: The Role of Electron Microscopy LUCIAN0 ZAMBONI Department of Pathology and Laboratory Medicine, UCLA School of Medicine and Harbor-UCLA Medical Center, Torrance, California 90509

KEY WORDS

Human sperm, Ultrastructure, Pathology

In this article, the major contributions of electron microscopy to the present unABSTRACT derstanding of the physiology and pathophysiology of the human spermatozoon are reviewed. The ultrastructural organization of sperm organelles playing a significant role for cell function and, therefore, for the reproductive process is described. Also, the major abnormalities and defects of the various organellar systems and how they impair the reproductive function andlor the viability of the cell are reviewed.

INTRODUCTION The behavior of the human gametes following their release from the gonads differs in the two sexes. Upon leaving the ovary, the oocyte enters the oviduct where i t undergoes subtle, albeit important, maturative changes that enhance its receptivity to the spermatozoon and its fertilizability (Zamboni et al., 1965; Zamboni, 1972); it has a short functional life span (Adams and Chang, 1962) and unless it is fertilized within a few hours, the oocyte senesces, undergoes structural deterioration, and dies (Longo, 1974). Upon leaving the seminiferous tubules, the spermatozoa pass into and through the vasa efferentia and then enter the epididymis. The transit through the various segments of the epididymis is very important for sperm function; normally, in fact, it is in the epididymis that the male gamete sheds its no longer functional cytoplasmic remnants, the cytoplasmic droplet, acquires effective, forward progressive motility, and undergoes metabolic and chemico-physical modifications especially of the nucleus, plasma membrane, and cell surface that bring it closer to the acquisition of a full functional competence (for reviews of epididymal sperm maturation, see Austin, 1985; Bedford, 1975; Olson and Orgebin-Crist, 1982; Voglmayr, 1975). Also, the spermatozoa can spend relatively long periods of time outside the testes in the male as well a s female reproductive tract without apparent decrease in viability (Ahlgreen et al., 1975; Thibault, 1973). In contrast to the woman who generally contributes only one oocyte to the reproductive process, the normal man contributes hundreds of millions of spermatozoa even though access to the oocyte is restricted to one. Finally, the sperm has another unique requirement: its transit through the female reproductive tract is necessary not only to reach the oocyte but also to attain full reproductive competence through a process described for the first time simultaneously but independently by Austin (1951) and Chang (1951) and referred to as “capacitation” (vide infra). Consideration of the factors responsible for these dif-

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ferences between gametes would be beyond the scope of this review. One of these differences, however, i.e., the very large numbers of spermatozoa that are produced and contributed to the reproductive process as opposed to the single oocyte, is pertinent to the topic of this review. In fact, it is not unreasonable to speculate that, considering the physiologic heterogeneity of the sperm population in the ejaculate (Bishop, 1964) and the anatomic and functional obstacles disseminated along the path that the fertilizing sperm must travel to approach the egg, i t is only by producing and releasing high numbers of spermatozoa that a positive outcome of the reproductive process is rendered less improbable. Unlike the oocyte which waits for the arrival of the male gamete in the ampullar segment of the oviduct, the fertilizing spermatozoon is required to successfully perform multiple and highly complex tasks prior to fulfilling its reproductive function. It must be capable of vigorous and effective motility, must acquire penetrating capacity, fusogenic ability and fertilizing competence, and must contribute to the oocyte a normal genomic complement necessary for the assembly and subsequent development of the embryo. For each of these requirements, as well as for its own metabolism, the spermatozoon is served by specialized organellar systems (Fig. 1) that are structurally assembled in the course of its differentiation in the seminiferous tubules (spermiogenesis) but, as stated already, become functionally activated only outside the testis. None of these organelles, the organizational integrity and the functional effectiveness of which are so pivotal for sperm function and thus for the reproductive process, is visualizeable by light or phase contrast microscopy (at least not to sufficient degrees of definition), nor can they be easily and accurately evaluated by other methods of morphologic investigation such as cytochemical and immunocytochemical procedures. Due to their organelReceived March 1, 1990; accepted in revised form March 25, 1990. Address reprint requests to Luciano Zamboni, MD, Professor of Pathology and Laboratory Medicine, UCLA School of Medicine, Harbor-UCLA Medical Center, 1000 W. Carson Street, Torrance, CA 90509.

ELECTRON MICROSCOPY OF HUMAN SPERM

Fig. 1. Low power electron micrograph of sperm in human semen. The sperm head (HI and the neck (N), midpiece (MP) and principal piece (PP) of the flagellum are indicated. x 12,500.

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Fig. 2. High power view of the structures in the neck segment of the sperm flagellum. C, capitulum; SC, segmented columns; PC, proximal centriole; N, nucleus. x 50,000. Fig. 3. Transverse section view of the proximal centriole. x 76,000.

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lar nature, they can be best studied only by transmission electron microscopy, a method which is thus key to basic investigations on sperm morphophysiology and extremely important also whenever in-depth assessment of the structural integrity and functional preparedness of the male gamete is clinically required. In the context of this review, I will survey the various sperm organellar systems, their morphogenesis, structural organization, functional role, and their pathology together with its limiting effect on sperm function. Excellent reviews of normal sperm ultrastructure have been prepared by Fawcett (19751, Olson (1982), Pedersen and Fawcett (1976), and Phillips (1975). Comprehensive surveys of normal spermato- and spermiogenesis are those by Holstein and Roosen-Runge (1981), Phillips (19741, and Zamboni (1971).

MATERIALS AND METHODS In the author’s laboratory, semen preparation for electron microscopic analysis is carried out as follows. The sample, collected by masturbation, is allowed to liquefy. One drop of the fresh, liquified sample is then placed on a microscopic slide under coverslip and examined to obtain a n overall impression of the cellularity of the sample, the motility of the spermatozoa, and their “gross” morphology. This preliminary evaluation is important not only to determine the adequacy of the specimen to be submitted to electron microscopy but also to establish a correlation between light microscopic and ultrastructural findings. Upon completion of the light microscopic evaluation, the sample is fixed, without separating the cellular fraction from the seminal plasma, in large volumes (20-25 ml) of picric acidformaldehyde (PAF) in phosphate buffer; this fixative, commonly referred to a s Zamboni’s fixative, was devised by Stefanini et al. (1967) specifically for the preservation of spermatozoa for ultrastructural analysis.’ After 30 minutes, the semenifixative admixture is gently centrifuged to constitute a pellet, the supernatant is decanted, and, after a couple of rinses in phosphate buffer, the pellet is post-fixed for 45-60 minutes in 1%OsO, in Verona1 buffer with salts added, dehydrated in increasing ethanol concentrations, subdivided into small fragments, and embedded in Epon 812. The blocks are first sectioned a t approximately 1p in thickness, and the sections, stained by flotation on 1% aqueous toluidine blue solution, are examined under a light microscope; upon identification of representative areas, the blocks are appropriately trimmed for ultrathin sectioning; after staining with lead hydroxide, the sections are examined with Hitachi H U l l E or 600 microscopes.

DISCUSSION Motility is a sperm function of highest relevance for reproduction. This is readily apparent if one considers that, physiologically, the sperm must travel a distance of 12-15 cm separating the site of deposition a t coitus (the vagina) from the site of fertilization (the upper segment of the oviduct), and must also overcome the ‘Other primary fixatives of high osmularity, such as that of Ito and Karnovsky 11968),can be used with equally excellent results.

anatomical imperviousness of the female reproductive tract (the stricture of the utero-tuba1 junction, the anfractuosities of the tubal epithelium), antagonistic dynamic forces (the beating of the cilia of the tubal epithelium, the currents of the oviductal fluid), and other barriers (the cervical mucus) disseminated along its percourse. Together with capacitation and the acrosome reaction (vide infra), motility is also a necessary preliminary for fertilization; in fact, several studies have shown good correlation between the particularly vigorous type of motility (hyperactivatedl displayed by the spermatozoa in the proximity of the eggs and their ability to penetrate through the egg vestments, especially the zona pellucida (Burkman, 1984; Fleming and Yanagimachi, 1982; Katz and Yanagimachi, 1981; Yanagimachi, 1983). Responsible for the initiation, maintenance, and effectiveness of motility is the complex internal machinery of the flagellum. As defined by the presence of specific structures, the flagelum can be subdivided into four individual regions: following a cranio-caudal direction, they are the neck, the mid-piece, the principal piece, and the end piece. The main function of the elements of the neck is to maintain the connection between head and flagellum and coordinate their movements. They are the connecting piece, consisting of the capitulum and nine peripheral segmented columns, and the proximal centriole (Figs. 2 and 3). The capitulum is a nearly horizontal structure that lies against the implantation fossa on the posterior surface of the nucleus. The implantation fossa is lined by the basal plate and the actual connection between head and flagellum is mediated by a n articular structure consisting of a finely filamentous, proteinaceous material filling the narrow space between the basal plate and the capitulum. The segmented columns are regularly arranged around the circumference of the neck; cranially, they implant onto the capitulum, some individually and others after having fused together, while caudally their tapered extremities merge with the outer dense fibers of the flagellum. The connecting piece is made up of a fibrous protein with a repeating period of 665 within which there is a dense band measuring 520 in length and consisting of ten evenly spaced bands (Fawcett and Phillips, 1969; Phillips, 1975). Its chemical composition, periodicity, mode of assembly, and centriolar origin (vide infra) render the connecting piece analogous to the rootlets of cilia of epithelial cells, even though it does not have a n anchoring function as the latter do. The proximal centriole, the only one of the two original spermatid centrioles to be retained in the mature spermatozoon (vide infra), occupies a central position in the most cranial portion of the neck just below the capitulum (Figs. 2 and 3); it is surrounded by the segmented columns and oriented with its long axis perpendicular to t h a t of the flagellum. It displays a typical organization and consists of a circular system of nine radially oriented triplet microtubules (Fig. 3); a t the internal extremity of the centriole, each triplet loses one element while the remaining two bend almost at right angles to continue with the nine sets of double microtubules at the periphery of the axonemal complex

a


(Zamboni and Stefanini, 1971). Its structural organization and its relationship with the axonemal microtubules render the proximal centrile analogous to the basal bodies of cilia except that the latter are oriented parallel to the ciliary shaft whereas the orientation of the centriole is perpendicular to the axis of the flagellum. The function of the proximal centriole has yet to be unequivocably defined. Due to its similarities with ciliary basal bodies, Zamboni and Stefanini (1971) postulated that it could be considered a s the element responsible for the initiation of flagellar movements; however, the validity of this hypothesis is significantly diminished by the absence of the proximal centriole in spermatozoa of a few mammalian species (Woolley and Fawcett, 1973). The structures of the connecting piece become gradually assembled during the spermatidic stage of spermiogenesis beginning with the migration of the two centrioles to the “flagellar” pole of the spermatid nucleus (Fig. 4). The centriole situated close to the nucleus is referred to as proximal and the other, oriented with its long axis perpendicular to the former and in the same direction of the developing flagellum, as distal. Detailed ultrastructural studies by Fawcett and Phillips (1969). Phillips (1974); Woolley and Fawcett (19731, and Zamboni and Stefanini (19711, have clearly shown that the two centrioles directly contribute to the assembly of the connecting piece and axonemal microtubules of the definitive spermatozoon. Both centrioles participate in the morphogenesis of the capitulum and the segmented columns. The distal centriole is the site of origin and the template of the doublet microtubules of the flagellum axoneme which originate from the ends of the A and B microtubules of each centriolar triplet growing gradually in length by polymerization of tubulin at their distal extremities (Fig. 5). The proximal centriole is instead involved in the formation of the centriolar adjunct (Figs. 5 and 6). This is a transient neck structure of still unknown function (Fawcett and Phillips, 1969; Holstein and Roosen-Runge, 1981) which is organizationally similar, but not identical, to the centriole proper; it usually disappears prior to spermiation but, in man, can be found occasionally in ejaculated spermatozoa. In general, any structural abnormality of the connecting piece results in defective motility, therefore limiting or nullifying the ability of the spermatozoon to reach the site of fertilization. The two most frequent of these defects are the eccentric implantation of the connecting piece with respect to the sperm head (Fig. 5) and the abnormal topographic relationship between capitulum and segmented columns, both causing the head to become flexed over, rather than aligned with, the axis of the flagellum (Fig. 7). The observation that these defects are associated with dyskinetic motility corroborates the notion that a n important function of the connecting piece is to harmoniously coordinate head and flagellar movements a s prerequisite for effective motility. The most severe pathologic condition of the neck is the separation of the head from the flagellum (Figs. 8 and 9), a defect that is referred to a s decapitation (Luders, 1976; Perotti et al., 1981). The defect is congenital

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(Baccetti et al., 1989a) and appears to originate from either abnormal chemical composition of the filamentous material filling the space separating the capitulum from the basal plate (LeLannou, 1979) or abnormal position of the centrioles with respect to the spermatid nucleus a t the time of flagellar development (Baccetti et al., 1984; Chemes et al., 1987b). In most cases, the defect becomes manifested in the epididymis simultaneously with the acquisition of motility when the vigorous movements of the flagellum overpower the weak or otherwise defective connection between the two main components of the cell bringing about their separation. The detached heads mostly remain in the epididymis where they are likely disposed of by macrophages as is the case in farm animals and nonhuman primates (Roussel et al., 1967), the headless flagella, which beat vigorously since they are provided with a n intact motility apparatus, coming to represent the sole or prevailing constituent of the ejaculate. The presence of this defect, which is obviously incompatible with any fertility potential, can be determined exclusively by electron microscopy; it is difficult, in fact, to identify this abnormality a t conventional semen analysis because the most cranial segment of the headless flagella are invariably enveloped by a cytoplasmic droplet (Figs. 8 and 9) that appears a s a small head a t the light microscope (Zamboni, 1987a,b); hence the misnomer “pin-head defect” (Zaneveld and Polakoski, 19771, a misnomer that might result in the underestimating of the consequences of this abnormality. In fact, some authors still consider the condition to be deprived of biologic importance (Bergman et al., 1982). In men, the defect is not as frequent as in farm animals (for a review of the pertinent literature, see Zamboni, 1987a,b), where it constitutes a n irreversible cause of sterility as well. The elements of the axonemal complex extend from the proximal centriole to the end of the flagellum. They are typically organized in a 9 + 2 pattern: transverse sections through the flagellum (Fig. 10) show nine sets of double microtubules circumferentially arranged at the periphery of the axonemal complex and two single microtubules in the center, a n organization that is characteristic of flagella and cilia of all eukaryotic organisms (Gibbons, 1981). Each peripheral doublet consists of a subunit A which is a complete microtubule and a subunit B which is incomplete and attached with its extremities to the wall of A. Each subunit A and the two microtubules in the center of the axonemal complex consist of 13 heterodymers (aand p) of tubulin, a structural protein of about 110,000 daltons in molecular weight; only 10 heterodymers make up the subunits B of each peripheral doublet. If cross-sectioned sperm flagella are examined a t high magnification, several accessory elements imposing physical constraints on the axoneme and contributing to the generation and maintenance of motility are visualized (Fig. 10). Departing from each subunit A and extending with slightly diverging courses towards subunit B of the next doublet in clockwise direction are two linear structures, the dynein arms, distinguished, due to their position, into inner and outer; they are regularly spaced along the extension of the A subunit at 24 nm

Fig. 4. Proximal (PC) and distal centriole (DC) at one pole of a spermatid nucleus in an early stage of flagellar development. x 18,000. Fig. 5. Later stage of development of the flagellum than that illustrated in Figure 4. The relationship of the distal centriole (DC) with the axonemal microtubules of the elongating flagellum, and that of the proximal centriole (PC) with the centriolar adjunct (CAI, are evident. Notice the eccentric implantation of the neck on the sperma-

tid nucleus (compare with Fig. 4) which is the origin of the “flexed head’ defect shown in Figure 7. x 32,000. Fig. 6. Centriolar adjunct (CA) extending from the proximal centriole (PC) in late spermatid stage of spermiogenesis. x 19,500. Fig. 7. “Flexed head’ defect in mature spermatozoon. The defect is caused by eccentric implantation of the elements of the neck on the spermatid nucleus at the onset of flagellar development (see Fig. 5). x 24,000.


Figs. 8, 9. Headless flagella in the seminal fluids of individuals with the “decapitation” defect. At light microscopic examination, the cytoplasmic droplets surrounding the proximal segments of the headless flagella are frequently interpreted as small sperm heads (see text). Figure 8, x 27,000; Figure 9, x 21,000.

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Fig. 10. Normal ultrastructural organization of the axonemal complex, dense outer fibers, and mitochondria in the midpiece of a sperm flagellum sectioned along a transverse plane. x 85,000.

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intervals, and are made up of dynein, a protein possessing magnesium-dependent adenosine triphosphatase (ATPase) activity. Surrounding the two central single microtubules is a ring-like structure, the helical sheath, towards which converge other linear densities, the radial spokes, each departing from subunit A of each peripheral doublet. Finally, the axoneme-associated structures include the nexin links, proteinaceous elements seen as linear densities extending between adjacent doublet microtubules and thought to be responsible for the maintenance of the cylindrical configuration of the other elements of the axoneme. As already mentioned, the assembly of the axonemal complex occurs during the spermatidic stage of spermiogenesis, the gradual elongation of its peripheral microtubules being brought about by continuous polymerization of tubulin, their constituent protein, from the microtubules of the distal centriole (Figs. 4 and 5). Throughout the midpiece and for a short segment of the principal piece, the 9 + 2 system of axonemal microtubules is surrounded by nine outer dense fibers (Fig. lo), each connecting cranially with the distal extremities of the segmented columns of the connecting piece (Fig. 2). The presence of these fibers results in the 2 organization of the flagellum typical of 9 + 9 spermatozoa of animals with internal fertilization. The size and the shape of the fibers are species characteristic, those of the human sperm being the smallest of the phyla. The material of the fibers is keratinous; it contains proteins rich in cysteine and proline and high numbers of disulfide bonds (Baccetti, 1982; Olson and Sammons, 1980). The assembly of the outer dense fibers occurs slowly during spermiogenesis; in the rat, it is a multistep procedure that evolves from step 8 to step 19, proceeds gradually in a proximal-to-distal direction and is accom anied by significant incorporation of 'H-proline and H-cysteine (Irons and Clermont, 1982a). In the midpiece, the axonemal microtubules and the outer dense fibers are ensheathed by mitochondria (Fig. 10) in helicoidal arrangement (Fig. 11);the helix of human spermatozoa has 11-13 gyres (Fig. 11)with two mitochondria per gyre (Fig. 10). The organization of the mitochondrial sheath, the only structure unique to the midpiece, becomes established gradually during the spermatidic stage of differentiation as a result of the migration of the mitochondria from the various areas of the cytoplasm to the region where the flagellum is being organized (Figs. 12 and 13). Those mitochondria that have not entered in the composition of the helix at the end of the process remain scattered in the no longer functional portion of cytoplasm, the cytoplasmic droplet, surrounding the head and the initial segments of the flagellum of newly formed spermatozoa. By the time the droplets are shed during sperm transit through the epididymis where they are phagocytized by the epithelial cells (Hermo et al., 19881, the midpiece mitochondria remain the sole representatives of this class of organelles in mature spermatozoa.2 Re-

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"In man, this does not occur regularly: presence of spermatozoa still possessing voluminous cytoplasmic droplets is not a t all unusual in the human semen.

cently, Olson and Winfrey (1986) demonstrated the presence of a reticular network of electron-dense material adherent to the mid-piece mitochondria; these authors proposed that this network, referred to a s submitochondrial lattice or reticulum, has a cytoskeletal function and plays a role in directing the precise spatial arrangement of the mitochondria around the circumference of the mid-piece (Olson and Winfrey, 1986; Olson et al., 1987). Beginning a t the annulum, the ring-like structure demarcating the transition from the mid- to the prin2 system of the axonemal cipal piece, the 9 + 9 microtubules and outer dense fibers become surrounded by the fibrous sheath which extends for the whole length of the principal piece. The fibrous sheath consists of: a) two longitudinal columns (Fig. 15) running along opposite sides of the flagellum and lying on the same plane of the two microtubules in the center of the axonemal complex, and b) a cranio-caudal array of regularly spaced circumferential ribs (Fig. 14) oriented perpendicular to the axis of the columns, each surrounding one half of the circumference of the flagellum and attaching to the columns with their extremities (Fig. 15). Biochemical analysis of fibrous sheath material of rat spermatozoa has shown that it consists of several polypeptides with different molecular weights, the prevailing one having a molecular weight of 80,000 daltons and appearing to be a phosphoprotein (Brito et al., 1989; Olson, 1979). The fibrous sheath becomes assembled in late stages of spermiogenesis. The columns become organized slowly, their assembly in rat spermatids having been estimated to occur over a 15 day period; they first appear at the distal end of the flagellum in step 2 and extend along a caudo-cranial direction to reach the annulum in steps 17123 (Irons and Clermont, 1982b). The assembly of the ribs is instead much more rapid (4-5 days) and occurs between steps 11 and 15 (Irons and Clermont, 1982b). They appear to originate from the spindle-shaped body (Figs. 16 and 17) seen in human spermatids as a fusiform structure consisting of concentric arrays of spirally oriented microtubules surrounding the outer dense fibers and continuing with the ribs of the developing fibrous sheath (Fig. 17; Holstein and Roosen-Runge, 1981; Wartenberg and Holstein, 1975).The spindle-shaped body is a transitory structure which is all but gone a t spermiation but can be seen sometimes in human ejaculated spermatozoa. Each of these flagellar elements plays a key role in allowing the spermatozoon to move effectively in a forwardly direction. It has been known now for more than 30 years as result of original observations made by electron microscopy (Afzelius, 1959) that flagellar movements do not, as originally thought, result from conformational changes of the molecules of contractile proteins resulting in shortening and lengthening of the axonemal microtubules, but rather from the sliding of the microtubules alongside one another in a manner that is strikingly similar, from a mechanical viewpoint, to the sliding motion of actin and myosin filaments of skeletal and myocardial muscles during the contraction-relaxation cycle (Haimo and Rosenbaum, 1981; Linck, 1979; Satir, 1979). Essential for the slid-

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Fig, 11. The helicoidal organization of the midpiece mitochondria is evident in this micrograph. X 18,500. Figs. 12, 13. Two phases of spermiogenesis leading to the final organization of the midpiece mitochondria as shown in Figure 11. In

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a n initial phase (Fig. 12), the mitochondria are still scattered throughout the cytoplasm of the early spermatid. In a late spermatid (Fig. 131, they line up alongside the initial portion of the developing flagellum. Figure 12, X 7,000; Figure 13, X 26,000.

Figs. 14, 15, The ultrastructural organization of the elements of the fibrous sheath as seen in longitudinally (Fig. 14) and transversely sectioned (Fig. 15) principal pieces. Figure 14, x 12,500; Figure 15, x 80,000. Figs. 16,17. These micrographs illustrate the spindle-shaped body usually seen around developing flagella of late spermatids. The con-

centrically organized microtubules of the spindle-shaped body are throught to be the templates of the hemispherical ribs of the fibrous sheath even though the morphogenetic mechanisms remains to be elucidated. The close relationship between spindle-shaped body microtubules and the ribs of the fibrous sheath is evident in Figure 17 (arrowheads). Figure 16, X 22,000; Figure 17, X 16,000.


ing activity of the axonemal microtubules is the presence of ATP synthesized by the mitochondria of the flagellar midpiece which, in the presence of Mg2+ and appropriate pH and ionic environment, is hydrolyzed by the ATPase of the dynein arms. The function of the dynein arms is thus similar t o that of the meromyosin filaments bridging the spaces between actin and myosin filaments of the striated muscle. The localization of the mitochondria in the immediate proximity of the axonemal microtubules is obviously ideal for making available to the latter the chemical energy required for their mechanical activity. The function of the dense outer fibers is less clear. Considering the chemical composition of their constituent material (see above) as well as the fact that their amino acid profile differs from that of contractile proteins (Olson and Sammons, 1980), the fibers may not serve a contractile function but may instead be elastic stiffening structures (Fawcett, 19771, a hypothesis reinforced by the observation that spermatozoa of animals with large outer dense fibers have a stiffer beat than that of sperm with fibers of a smaller size (Phillips, 1972). It has been proposed that their function may be to impart resilient rigidity to the flagellum (Swan et al., 1980) as well as provide protection to the delicate microtubules of the axoneme during sperm transit through male and female reproductive tracts (Zamboni, 198713). On the other hand, it has been reported (Feneux et al., 1985) that defects of the outer dense fibers result in abnormal motility (dyskinesia) and reduction of motility effectiveness; this would suggest a direct role of the fibers in motility. Perhaps both functions coexist. A scaffolding, purely skeletal role is classically attributed to the fibrous sheath, its flexibility, so necessary for the beating movements of the flagellum, being assured by the anatomical organization of its rib-like elements. Considering the importance of their function for the normalcy and effectiveness of sperm motility and considering how important both of these parameters are for reproduction, the demonstration that pathologic conditions of the flagellar elements regularly result in impairment of motility, albeit of different degrees, and consequently, in limitation of the sperm reproductive competence, should not come as a surprise. However, not all conditions characterized by ineffective motility result from structural abnormalities of the elements of the flagellum; on the contrary, in most cases they are due to decreased sperm viability or to sperm death. The ability to distinguish asthenozoospermic conditions due to sperm degeneration from those caused by structural defects of the flagellum is extremely important and not only from a diagnostic perspective. Whereas viable spermatozoa that are ineffectively motile or are immotile due to a flagellar pathology may be capable of fertilizing oocytes in vitro (Cohen et al., 1984, 1985; Palermo et al., 1989) and may be microsurgically introduced into the oocyte (Bongso et al., 19891, respectively, generalized necrozoospermia is incompatible with any reproductive potential under any circumstances. The ability to distinguish asthenozoospermias due to decreased sperm viability from those caused by the presence of flagellar defects is important also from another viewpoint. Conditions characterized by emis-

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sions of degenerating spermatozoa are frequently caused by an epididymal pathology (epididymal necrozoospermia). Wilton et al. (1988) have reported that in these situations sperm viability and motility can be improved by increasing the frequency of ejaculations and thus decreasing the length of sperm permanence in the epididymis. How can the presence of nonviable or dead spermatozoa in the ejaculate be determined? Several methods are available. The most common is the dye exclusion method (most commonly used is eosin Y) that permits the identification of degenerating or dead spermatozoa on a smear of seminal fluid due to their pink coloration resulting from the diffusion of the dye through their structurally damaged plasma membranes (Eliasson and Treichl, 1971). More recently, the hypo-osmotic swelling test (HOS) was introduced by Jeyendrau et al. (1984) to differentiate nonviable from viable human spermatozoa: exposed to hypo-osmotic conditions, the latter display curling and bulging of the flagella due to influx of fluids through their functionally integrous plasma membranes, in contrast to the former that fail t o exhibit such a response. An indirect approach to the assessment of sperm viability is to determine the condition of the acrosome by supravital and immunocytochemical staining methods (Cross et al., 1986; Talbot and Chacon, 1981) to identify spermatozoa that have lost this delicate organelle as a consequence of cell degeneration or death. These procedures vary in reliability and technical complexity, the easiest one being the dye-exclusion method. In any case, they are only rarely used in the clinical practice and their application is mostly restricted to research purposes. Consequently, just about any condition characterized by reduced sperm motility or immotility is labelled with the generic term “asthenozoospermia” and only exceptionally are attempts made to investigate its etiology, i.e. whether it is due to decreased sperm viability or flagellar pathology. This can be readily accomplished by electron microscopy, the only method that allows simultaneous assessment of sperm viability and flagellar organization. At the electron microscope, the subcellular changes characteristic of necrosis are readily identified (Wilton et al., 1988; Zamboni, 1987a,b). There is fragmentation and loss of significant portions of the plasma membrane (Figs. 18-20), swelling of the mitochondria and loss of the cristae (Fig. 181, karyolysis and karyorrhexis, and degenerative changes of the acrosome ranging from swelling, vesiculation of the acrosomal membranes and lack of matrical homogenity, to complete loss of the organelle (Figs. 18 and 19).3 Another frequent expression of sperm degeneration is the transformation of the axonemal microtubules into filaments (Fig. 20; Wilton et al., 1988; Zamboni, 1987a,b), a change that precedes their complete reabsorption. Generalized necrozoospermia is frequently accompanied by presence of inflammatory cells in the seminal fluid, prevalently neutrophils and histiocytes; often, both types of cells become engaged in the dis3Due to their similarity to those of the physiologic acrosome reaction (vide infra), these degenerative changes are referred to as the “false” or “spurious” acrosome reaction.

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Figs. 18, 19, 20. These micrographs illustrate the degenerative changes of the various sperm organelles in true necrozoospermic conditions. The regressive changes of the acrosome are shown in Figures

18 and 19, those of the mitochondria in Figure 18, and those of the axonemal microtubules in Figure 20 (for details, see text). Figure 18, x 18,500; Figure 19, x 12,000; Figure 20, x 40,000.


Fig. 21. Seminal fluid neutrophil in the process of internalizing necrotic sperm remnants. x 11,000. Fig. 22. Sperm-laden macrophage in seminal fluid.

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Fig. 23. Piospermia. The semen contains impressive numbers of sperm-laden macrophages as the one illustrated in the electron micrograph of Figure 22. 1-p,m thick, Toluidine blue stained plastic section. ~ 5 0 0 .

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posal of the necrotic spermatozoa by phagocytosis (Figs. 21 and 22). In the most extreme cases, true piospermic conditions become established (Fig. 23). Asthenozoospermic conditions without attendant sperm degeneration or agglutination require that attention be paid to the existence of flagellar defects; the most widely known of these are the abnormalities of the 9 + 2 system of axonemal microtubules. These defects cover a broad spectrum (for recent reviews, see Zamboni, 1987a,b) and include: numerical aberrations in excess or, most frequently, in defect (Figs. 24 and 25), ectopic localization of one or more microtubules, a n abnormality that frequently occurs in association with numerical aberrations (Fig. 25), and structural defects of the delicate elements associated with the axonemal microtubules, the dynein arms in particular (Fig. 26). To be diagnostically significant, any of these flagellar defects must be present in the totality or, a t least, the vast majority of the spermatozoa, the occasional occurrence of one or more of them being a feature of any sperm population (Hunter and deKretzer, 1966; Pryor et al., 1981; Zamboni, 1987a,b). Most ominous and function-limiting are: 1)the deletion of the two microtubules in the center of the axoneme; this may occur with a n intact system of nine peripheral microtubules (Fig. 24), the so-called 9 + 0 defect (Baccetti et al., 1979; Nistal et al., 1979; Zamboni; 1987a,b), or in association with numerical aberrations andlor ectopic localization of the latter (Fig. 25); 2) the absence of all axonemal microtubules (Baccetti et al., 1980; Zamboni, 1987b); and 31, the absence of one or both dynein arms from the doublet microtubules a t the periphery of the axonemal complex (Fig. 261, a defect that was first documented by Afzelius et al. (1975) and, since then, repeatedly described as frequent cause of sperm immotility (for a recent review, see Zamboni, 198713). The infertility of individuals with these and other axonema1 defects is clearly due to the inability of their sperm to reach the site of fertilization: it has been shown in fact that such immotile or dyskinetic spermatozoa can be otherwise capacitated, undergo the acrosome reaction, and penetrate the zona-free hamster oocytes (Aitken et al., 1983; Moryan et al., 1986). In some individuals, defects identical to those affecting the axonemal microtubules of the flagellum occur also in the axonemal microtubules of the cilia of ciliated epithelia thus becoming part and parcel of the pathologic constellation of the immotile or dyskinetic cilia syndrome first documented by Afzelius (1976). This association, that vividly demonstrates the congenital nature of these abnormalities (Eliasson et al., 19771, involves frequently, but not exclusively, the lack of the dynein arms (Fig. 27). In a minority of these individuals, the infertility caused by sperm immotility and the upper respiratory tract pathology resulting from ciliary immotility are associated with situs viscerum inversus and constitute the triad of the syndrome first described by Kartagener (1933) and known since his name. Cases in which the sperm immotility resulting from axonemal defects of the flagellum did not cause infertility have been occasionally reported (for a review, see Zamboni, 198713) but are rare.

A much less known flagellar defect, also of a congenital nature and resulting in total immotility and therefore in sterility, is the absence of the mitochondria in the midpiece (Figs. 28-30). The existence of this condition had been mentioned but briefly in the context of reviews of a variety of sperm abnormalities resulting in infertility or subfertility (Bartoov et al., 1980; deKretzer and Holstein, 1976; Ross et al., 1973; Zamboni 1987a). The defect occurs in two main varieties. The spermatozoa may possess a midpiece which obviously lacks the mitochondria (Fig. 291, or they may lack the midpiece altogether; in this case, the fibrous sheath implants directly onto the neck (Figs. 28 and 30). In either variety, the absence of the mitochondria results in the absence of the thick midpiece segment and, thus, in uniform thinness of the flagella. This peculiarity should be picked up at conventional semen analysis, except that its detection is often prevented by the presence of cytoplasmic droplets around the initial portions of the flagella (Figs. 28-30); thus, electron microscopy is the sole method available for the identification of this abnormality. In addition to preventing motility, the lack of mitochondria obviously has a deleterious effect on sperm viability: this is in fact demonstrated by the observation that spermatozoa without mitochondria are often degenerating (Fig. 30). The origin of the defect was traced electron microscopically by Holstein (1975) in late spermatids in testicular biopsies of patients with different andrologic disorders. Since it is difficult to imagine absence of mitochondria in spermatid precursors (such a n abnormality would be incompatible with cell replication and differentiation), one must conclude that, a s originally proposed by Ross et al. (1973), the defect very likely results from disorders of the internal remodeling processes that accompany the organization of the flagellum a t the spermatidic stage of spermiogenesis when the mitochondria, from their original cytoplasmic sites, become polarized around the centriolar region and the developing axonema1 elements departing from it (vide supra). It is probable that these abnormal patterns off lagellar development result from the pathology of the already described submitochondrial lattice or reticulum (Olson and Winfrey, 1986). This hypothesis reinforces the view that the final organization of the sperm flagellum, just like that of the head (vide infra), is the product of the precise work of a complex cytoskeletal machinery reaching a functional climax in the late spermatidic stage and the pathology of which, a fascinating subject to study, leads to sperm defects such a s the one just described. Another flagellar defect recently described by Chemes et al. (1987a) in five infertile patients with generalized immotility or severe asthenospermia (two were brothers) is the dysplasia of the fibrous sheath characterized by hyperplasia and disorganization of its elements. This defect unquestionably demonstrates that, albeit a scaffolding element, the fibrous sheath plays a key caodjuvatory role in sperm motility. The main structures of the sperm head, the acrosome and the nucleus (Fig. 311, are directly involved in the egg penetration and fertilization phases of the reproductive process. The acrosome is a n organelle that

Figs. 24, 25, 26. These micrographs illustrate various abnormalities of the axonemal microtubules. The deletion of some axonemal microtubules is shown in Figures 24 and 25: in Figure 24, the defect affects only the central microtubular pair (the 9 + 0 defect), whereas the sperm flagellum in Figure 25 additionally lacks one set of periphera1 doublets and has another set in ectopic position. The absence of the dynein arms is illustrated in Figure 26. Figure 24, x 55,000; Figure 25, x 55,000; Figure 26, x 47,000.

Fig. 27. Absence of dynein arms from the peripheral microtubular doublets of ciliary axonemal complexes. x 60,000. Fig. 28. Absence of mitochondria and midpiece: the principal piece, identifiable due to presence of the fibrous sheath, implants directly on the neck (see also Fig. 30). x 15,000.

Fig. 29. A different variety of a mitochondria-less spermatozoon. The sperm has a thin midpiece devoid of mitochondria and a normal positioning of the principal piece. x 12,000. Fig. 30. The absence of mitochondria results in decreased sperm viability as demonstrated by the presence of degenerative acrosomal changes similar to those shown in Figure 19. Initial regressive

changes of the acrosome are visible in the spermatozoon in Figure 29. x 14,000. Fig. 31. Head of normal spermatozoon showing the organization of the acrosome and nucleus (for details, see text). ES, equatorial segment of the acrosome. x 40,000.


forms directly from the Golgi complex in the early spermatidic stage of spermiogenesis in close association with the spermatid nucleus, a relationship that persists unchanged throughout the remodelling processes that both organelles undergo t o reach their definitive organization. The acrosome is limited by Golgi-derived membranes and its matrix is a product of the synthetic activity of the Golgi complex. Its morphogenesis (for reviews see Holstein and Roosen-Runge, 1981;Phillips, 1974; Zamboni, 1971) initiates at the time the prominent Golgi apparatus of the spermatid becomes positioned next to one pole of the nucleus, still spherical at this stage; one of the multiple vesicles situated on the concave face of the Golgi complex enlarges and becomes closely apposed against the nuclear envelope (Fig. 32). This vesicle, referred to as pro-acrosomal, continues to augment as result of other Golgi vesicles becoming confluent with it and contributing to it their membranes and internal spaces. The template of the acrosomal matrix is the pro-acrosomal granule (Fig. 32), a mass of electron dense material which is synthesized in the cavity of the original pro-acrosomal vesicle and increases progressively in volume by accretion as result of the addition of the content of other Golgi vesicles. Close to or at the time the dense material of the original granule fills the entire space of the pro-acrosomal vesicle, the nucleus of the spermatid begins to elongate and the pro-acrosomal vesicle, closely apposed against it, follows its elongation becoming crescentshaped and stretched around the one-half or the threequarters of the nucleus farthest from the centriolar pole (Fig. 33). Even though the issue has not been definitely resolved and is still controversial, it is likely that the shape modifications of the nucleus and acrosome, which occur simultaneously and in parallel, are both the result of tractional forces exerted by shrouds of microtubules altogether constituting the “manchette” or “caudal sheath” and coronally arranged around the nucleus and the acrosome (Figs. 34 and 3 3 , and bundles of cytoskeletal filaments in the Sertoli cell cytoplasm surrounding the spermatid. This hypothesis makes it easy to explain why, as it will be shown later, abnormalities of one of the two head components are frequently, if not regularly, accompanied by structural defects of the other (Bartoov et al., 1980; Zamboni, 1987a,b). In fully differentiated human spermatozoa, the acrosome covers the anterior four-fifths of the nucleus (Fig. 31); it is the cell’s most rostra1 component and thus the most advanced along the path of the cell, a position that is of utmost functional importance. It is of uniform thickness throughout except posteriorly where it becomes abruptly thinner (the equatorial segment). Under the electron microscope, the acrosomal matrix is homogeneous and highly electron opaque. The outer aspect of the acrosomal membrane is closely surrounded by the plasma membrane while the inner aspect runs parallel t o the envelope of the underlying nucleus. Because it contains several hydrolytic enzymes, some of which are of functional importance in enabling the spermatozoon to penetrate through the egg vestments (Bellve and O’Brien, 1983; Bhattacharyya and Zaneveld, 1982; Frazer, 1984; Harrison, 1983; Rogers

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and Bentwood, 1983; Tang et al., 1982; Tesarik et al., 19881,the acrosome is considered by some authors as a lysosome-like organelle (Allison and Hartree, 1970; Morton, 19761,whereas others (Friend, 1977; Harrison, 1983) view it as being more analogous to a secretory (zymogen)granule due to its participation in an exocytotic process at the time of the acrosome reaction: obviously, its derivation from the Golgi apparatus supports either hypothesis. The acrosome plays a key role in the reproductive process being functionally linked to both capacitation and the ability of the sperm to penetrate the egg vestments. It is now widely accepted (see the comprehensive reviews by Austin, 1985; Bedford, 1983; Chang, 1984; Rogers and Bentwood, 1983; Yanagimachi, 1983)that capacitation prepares the spermatozoon to undergo the structural changes of the acrosome reaction that are said to be necessary for its penetration through the egg vestments.* The acrosome reaction, first described by Austin and Bishop (1958), has been the topic of a plethora of studies focused on the mechanisms regulating male and female gamete interaction shortly before actual conjugation. In general, these studies have confirmed the early ultrastructural observations by Barros et al. (1967) showing that the structural changes of the acrosome reaction are multifocal fusions of the plasma membrane and the outer acrosomal membrane covering the acrosome region anterior to the equatorial segment, followed by breakdown of the fused membranes with consequent exocytosis of the organelle and extracellular liberation of its hydrolytic enzymes. Of these, hyaluronidase and acrosin, a trypsin-like enzyme, appear to play a key role in the digestion of the viscous matrix of the cumulus oophorus, which is especially rich in hyaluronic acid polymers, and the zona pellucida, respectively (for comprehensive reviews of the structural and functional aspects of the acrosome reaction, see Austin, 1985; Yanagimachi, 1983). For the sake of objectivity, however, it must be emphasized that, copious investigations notwithstanding, our comprehension of the functional role of the acrosome reaction in fertilization is still imprecise. Whether the reaction is important exclusively for the actual penetration of the sperm through the egg vestments or also for unmasking specific sperm surface components that recognize and bind to the zona pellucida (Saling, 1981), whether it represents the culmination of a series of endogenous changes in the spermatozoon elicited by, or allowed to occur as consequence of, the capacitation process (Bedford, 1982) or is induced by a substance emanating from the oocyte (Fraser, 1982, cited by Austin, 19851, and whether it occurs prior to or during sperm penetration of the cumulus oophorous or at the zona pellucida as indicated by recent evidence (Florman and Storey, 1982; Storey et al., 1984), are issues that remain to be resolved. Also under dispute is whether or not the acrosome reaction is important in bringing about modifications of the plasma membrane lining the equatorial segment of the acrosome and the post-acrosomal region of the head necessary to render the membrane fuso4As already mentioned, capacitation is instrumental also in enabling the spermatozoon to develop hyperactive motility.

Fig. 32-35. These micrographs depict sequential phases of acrosome formation and nuclear elongation during the spermatidic stage of spermiogenesis. The development of the proacrosomal vesicle (PAW from the prominent Golgi complex (GC) of an early spermatid is shown in Figure 32. PAG, proacrosomal granule. Figures 33 and 34 show successive phases of acrosome modeling in intermediate spermatids; notice also the gradual elongation of the nucleus and the

progressive aggregation of the chromatin. In the late spermatid in Figure 35, acrosome and nucleus have attained their nearly definitive shape and organizational characteristics. Notice the prominent arrays of the manchette (M) microtubules (visible also in Fig. 34 at arrowheads) anchoring all around the posterior extremity of the acrosome. Figure 32, )c 17,000; Figure 33, x 17,000; Figure 34, x 15,000; Figure 35, x 15,000.


genic with the egg plasma membrane as postulated by Yanagimachi (19811, and why the equatorial segment of the organelle is not involved in the acrosome reaction. Also unclear is the reason why spermatozoa exposed to denuded oocytes, such as those used for the sperm penetration assay, must be acrosome-reacted in order to be capable of penetration (Rogers, 1983). It is undeniable in any case that the organelle plays a pivotal role in the reproductive process as unequivocably demonstrated also by the fact that its pathology is a frequent cause of infertility (vide infra). Hence, the importance of being able to evaluate its structural integrity. The structural organization of the acrosome can be best and most directly demonstrated by electron microscopy. At light microscopic examination of fresh, unstained semen, the organelle is barely perceptible, its presence being discernible only because of the different luminosity displayed by the region of the nucleus covered by it. Phase microscopy reveals only a slightly increased amount of details and so do special procedures such as the already mentioned supra-vital and immunocytochemical staining procedures that were devised specifically for the demonstration of the organelle (Cross et al., 1986; Talbot and Chacon, 1981). For all practical purposes, thus, the acrosome generally escapes observation. This is highly unfortunate because, considering its role in reproduction, visualization of the acrosome is key for a precise assessment of the functional fitness of a given population of spermatozoa. The sperm nucleus appears at the electron microscope as a homogeneously compact mass of electrondense chromatin (Fig. 31) consisting primarily of a keratinoid body of heavily crosslinked deoxyribonucleic acid (DNA) coupled to basic proteins. The final composition of the mature sperm nucleus and its appearance are the result of salient chemical and physical changes that begin in relatively late stages of spermiogenesis and reach completion during epididymal transit (reviewed by Zamboni, 198713). They include elimination of the ribonucleic acid (RNA), replacement of somatic histones by protamines, and formation of chromatinstabilizing disulfide bonds. Simultaneously with nuclear elongation (Figs. 33-35), there is also gradual condensation of the chromatin which, from finely granular (early spermatid stage: Figs. 32 and 331, becomes aggregated at first into a coarsely granular pattern (late spermatid stage: Figs. 34 and 35) and then attains the homogeneous and highly compact degree of aggregation typical of the mature spermatozoon (Fig. 31). The degree of cross-linkage of the DNA of the sperm nucleus is so high as to render the chromatin strongly resistant to, for example, digestion by DNAse and mechanical disruption by sonication. Only strong agents such as detergents, acids, and lytic enzymes of bacterial origin (reviewed by Zamboni, 198713) can bring about partial decondensation of the chromatin. It has been justifiably postulated that such a high degree of unreactivity may serve to protect the genomic patrimony of the sperm from physical, chemical, or mutagenic insults during transit through the male and female reproductive tracts (Bustos-Obregon and Leiva,

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1983; Fawcett, 1981). As result of cleavage of disulfide bonds between protamine molecules and their proteolytic degradation, and the replacement of protamines with oocyte-derived histones (Longo, 19851, the sperm chromatin becomes instead readily decondensed in the ooplasm of the penetrated (activated) oocyte where it unravels in filamentous pattern and becomes highly hydrated as preliminary to the assembly of the male pronuc1eus.J Considering the functional importance of the nucleus for fertilization and for the assembly and development of the embryo, the ability to evaluate its structural organization is of importance for prospective assessment of semen quality. Yet, the methods that are routinely utilized to assess sperm morphology allow the visualization of the head but not that of the nucleus, especially because it is covered by the acrosome for the most part; at best, only its size and overall shape can thus be determined. In this case, too, transmission electron microscopy is the only method available for this purpose. The pathology of the acrosome can be classified into three main categories: acrosomal hypoplasia, structural defects, and absence of the organelle. Acrosomal hypoplasia (Figs. 36 and 37) is one of the most common pathologic conditions of the organelle and a frequent cause of infertility (Zamboni, 1987a,b). It is important to reiterate that without electron microscopy the infertility caused by this abnormality would remain unexplained since the defect cannot be detected at conventional semen analysis. At the electron microscope, the acrosomes appear considerably thinner than normal, the difference in thickness that normally distinguishes the bulk of the organelle from the equatorial segment is effaced, the matrix has a markedly decreased electron opacity, and the acrosomes are frequently separated from the nuclei (Figs. 36 and 37). The shape of the organelle is frequently distorted as well. In most cases, the defect is associated with nuclear abnormalities, especially shape deformations, presence of intranuclear vacuoles and inclusions (Fig. 361, and immaturity of the chromatin (Fig. 37), i.e., persistence ofthe coarse aggregational pattern typical of the chromatin of intermediate spermatids. The sterility caused by the defect is very likely linked to the impossibility of these defective spermatozoa to become properly capacitated, or to penetrate the egg vestments, or both, a hypothesis corroborated by the fact that they fail to penetrate the zona-free oocytes of the hamster or are capable only of minimal penetration coefficients. The defect is not degenerative in nature but, as indicated by the otherwise healthy appearance of the sperm and concomitant presence of nuclear abnormalities, is the expression of deranged spermiogenesis. One of the most frequent structural defects of the acrosome is the presence of inclusions almost invariably represented by clusters of vesicles and membranebound granules of different sizes (Fig. 38). Often, but 5Nuclear decondensation occurs also in the ooplasm of denuded hamster oocytes penetrated by human spermatozoa In the course of the sperm penetration assay This has led to the utilization of these decondensed nuclel for karyologlc studies aimed a t assessing the normalcy of human sperm chromosomes (Martln, 1985, Rudak et a1 , 1978)

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Figs. 36, 37. These micrographs illustrate spermatozoa with hypoplastic acrosomes. Not only are the organelles thinner than usual but they are also distorted in shape and separated from the nucleus. The frequent association of acrosomal hypoplasia and immaturity of the chromatin, characterized by aggregational patterns similar to those of spermatids (see Fig. 341, is shown in Figure 37; chromatin

immaturity often leads to nucleomalacia as shown. Figure 36, x 8,500; Figure 37, x 25,000. Fig. 38. Intra-acrosomal inclusions in spermatozoon in seminal fluid. x 33,000.


not regularly, the inclusion-bearing acrosomes have distorted shapes; in general, the volume of the organelle occupied by the matrix is significantly reduced as compared to normal, and matrical electron opacity is decreased. The defect, which may or may not be accompanied by abnormalities of the nucleus, does not appear to be of a degenerative nature since ultrastructural examination of testicular biopsies of individuals affected by this sperm abnormality discloses that intraacrosomal inclusions are present already in intermediate spermatids (Fig. 391, thus indicating that it is the expression of abnormal acrosome development. Very likely, the inclusions represent Golgi vesicles that failed to become incorporated in the developing organelle and remained sequestered as individual entities within the confines of the original pro-acrosomal vesicle. The absence of the acrosome can be either acquired or congenital. The first is always the expression of a degenerative process affecting the acrosomes just like other sperm components and including the already described changes eventuating in the dissolution of the organelle (Figs. 18, 19, and 30). The congenital variety is the agenesis of the acrosome (Figs. 40 and 411, a condition characterized by the production of spermatozoa lacking the organelle. This acrosomal defect is regularly accompanied by two other head abnormalities: sphericity of the nucleus, hence the misnomer “round head defect” frequently used to refer to the agenesis of the acrosome, and the already mentioned immaturity of the chromatin. This triad, documented in all cases reported in the literature (comprehensively reviewed by Zamboni, 1987b), provides additional, almost unequivocable evidence supporting the view that the morphogenesis of the two head components is brought about by commonly shared remodeling mechanisms. The defect has a familial trait (Florke-Gerloff et al., 1984; Kullander and Rousing, 1975; Nistal et al., 1978; Zamboni, 1987a) and appears to be transmitted polygenically (Florke-Gerloff et al., 1984). Its morphogenesis, traced in testicular biopsies primarily by electron microscopy but also by histochemical and immunohistochemical methods (for a review, see Zamboni 1987b), appears to result from a combination of factors that include abnormal topographic relation between the Golgi complex and the nucleus of the spermatid, abnormal or defective secretory activity of the Golgi complex, and structural and functional defects of the microtubules of the manchette. Obviously, the defect is incompatible with any fertility potential, under physiologic conditions or otherwise, and results from the impossibility of the spermatozoa to be capacitated and to penetrate the oocytes (Weissberg et al., 1982). The most frequent nuclear abnormalities are the immaturity of the chromatin and the presence of intranuclear vacuoles and inclusions. The first has been already mentioned in conjunction with acrosome hypoplasia (Fig. 37) and agenesis (Figs. 40 and 41). It is important t o add here that, in addition to being a cofactor together with the pathology of the acrosome in limiting the functional competence of the sperm, nuclear immaturity per se very likely is capable of preventing fertility. It has been determined, in fact, that

43 1

the DNA of spermatozoa with only partial condensation of the chromatin is single-stranded rather than double-stranded (Evenson et al., 1980; Pedersen, 1987) and, consequently, more prone to undergo denaturation as indicated also by the tendency of the immature chromatin to unravel in coarse strands or filamentous patterns (Fig. 37). Moreover, nuclei of spermatozoa of men with “idiopathic” infertility have often been found to possess somatic histones instead of protamines (Silvestroni and Frajese, 1978; Silvestroni et al., 1976); from this it can be inferred that the chromatin of these spermatozoa is probably immature since it has been reported (cited by Zamboni, 1987b) that the chemical composition of sperm nuclei with immaturity of the chromatin reveals failure of somatic histones to be replaced by basic proteins. Finally, the reported association of immaturity of the chromatin and chromosomal abnormalities (Kjessler, 1974; Abramsson et al., 1982) corroborates the view that the defect is in itself a cause of infertility, or may have teratologic consequences. The presence of spermatozoa exhibiting intranuclear vacuoles may be occasionally noted in the semen of men of proven fertility; in these cases, the vacuoles are usually single and of a small size. The nuclear defect being reviewed here is instead characterized by very large, often multiple, and confluent vacuoles (Fig. 42), the presence of which results in drastic reduction of the chromatin and profound deformations of the shape of the sperm head. A wide variety of sequestered material is usually found in the vacuoles, ranging from cytoplasmic organelles to arrays of membranes to lipid droplets. Usually, the nuclear vacuoles and inclusions occur in association with other nuclear defects, especially immaturity of the chromatin, and hypoplasia and shape deformation of the acrosome (Fig. 42). This pathology subtends the existence of abnormal processes of nuclear elongation and chromatin condensation. Electron microscopic studies performed on testicular biopsies of individuals with this defect, in fact, regularly demonstrate the presence of voids (vacuoles) or cytoplasmic organelles (inclusions) in the context of early and late spermatid nuclei undergoing elongation (Fig. 43); similar observations were made by Holstein (1975) in an extensive ultrastructural study of testicular biopsies from a large series of subfertile or infertile patients with a variety of andrological disorders, and by Holstein and Schirren (1979) in a comprehensive review of abnormal patterns of spermatid differentiation. The last example of abnormalities involving both nucleus and acrosome is the “crater defect,” a malformation of the sperm head characterized by a profound, crater-like depression of the nucleus and corresponding distortion of the acrosome, the shape of which adapts to the abnormal nuclear outline (Fig. 44); the acrosomes of spermatozoa with this defects are invariably hypoplastic. The abnormality was reported for the first time recently by Baccetti et al. (1989b) in an infertile man, but its occurrence in farm animals where it is associated with reduced fertility had already been documented in numerous studies (reviewed by Johnson and Hurtgen, 1985). Electron microscopic evaluation of testicular biopsies of patients with this abnormality discloses that the defect develops in very early stages of

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Fig. 39. The genesis of intra-acrosomal inclusions similar to those shown in Figure 38 is seen in a spermatid at an intermediate stage of acrosome development. x 30,000.

Figs. 40, 41. Agenesis of the acrosome: notice the association of the defect with sphericity of the nucleus and immaturity of the chromatin (for details, see text). Figure 40, x 85,000; Figure 41, x 30,000.


Figs. 42, 43. Intranuclear inclusions in a mature spermatozoon (Fig. 42) and the genesis of the defect in a late stage spermatid (Fig. 43). Notice the pleomorphic variety of the intranuclearly sequestered cytoplasmic elements, the deformed nuclear shape, the marked reduction of the chromatin, and the hypoplasia and abnormal shape of the acrosome. Figure 42, x 22,000; Figure 43, x 24,000.

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Figs. 44, 45. “Crater” defect in the nucleus of a late spermatid (Fig. 44) and the genesis of the defect in an early spermatid (Fig. 45); the abnormality appears to originate from overdistension of the proacrosomal vesicle which depresses the nuclear profile and induces it to assume a C-like shape. Figure 44, x 18,000; Figure 45, x 13,000.

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acrosome formation, a t the time the pro-acrosomal ves- considers that the positive outcome of the latter rests icle first appears, and that it is caused by a n abnor- primarily on the successful encounter and interaction mally large and overdistended pro-acrosomal vesicle of two isolated cells, the sperm and the egg, and that which, rather than adapting its shape to the convexity these are in turn predicated upon the normalcy of the of the spherical profile of the spermatid nucleus, de- cells’ organellar systems. By definition, the latter can presses it thus inducing the nucleus to assume a C-like be evaluated only by transmission electron microscopy. shape (Fig. 45). This morphogenetic pattern, already While this method of morphologic investigation cannot described by Holstein and Schirren (19791, suggests be applied to the study of oocytes for evident reasons, it also that this abnormality is the expression of the pa- certainly can be easily utilized for spermatozoa. Conthology of the cytoskeletal elements and forces respon- sidering the diagnostic and prognostic importance of sible for the final shape of the sperm head. However, the method, any lingering resistance to submit semen our observations indicate that it may not be the nu- to electron microscopic evaluation whenever indicated clear molding that influences the shape of the ac- can not longer be justified. rosome as hypothesized by Baccetti et al. (1989b); rather, it would appear that the pathologic condition of REFERENCES the nucleus is brought about by abnormal acrosome Abramsson, L., Beckman, G., Duchek, M. and Nordenson, I. (1982) development. The infertility of men with the crater deChromosomal aberrations and male infertility. J. Urol. 128:52-53. fect (Baccetti et al., 198910; Zamboni, unpublished) is Adams, C.E., and Chang, M.C. (1962) The effect of delayed mating on fertilization in the rabbit. J. Exp. 2001. 151:155-161. probably linked to chromatinicichromosomal abnorAfzelius, B.A. (1959) Electron microscopy of the sperm tail: Results malities as well a s hypoplasia of the acrosome. obtained with a new fixative. J. Biophys. Biochem Cytol. 5:269-

CONCLUSIONS I trust that this review, albeit necessarily cursory and incomplete, has convincingly demonstrated how important the contribution of electron microscopy has been for our understanding of basic sperm function and how essential the ultrastructural analysis of the human semen is for a precise assessment of the structural integrity of the spermatozoa as basis of their functional fitness. The various clinical situations in which the ultrastructural examination of the semen is either necessary or strongly indicated have been recently reviewed by the author (Zamboni, 1987a,b). The most obvious are unexplained infertile conditions (so-called idiopathic infertility), whenever there is a discrepancy between semen quality assessed to be within normal limits by conventional methods of semen analyses and: a), negative reproductive outcome in the absence of a female factor, and b), negative zona-free hamster oocyte sperm penetration assay (Yanagimachi et al., 1976), and conditions characterized by generalized sperm immotility (asthenozoospermia), where it is diagnostically, prognostically, and therapeutically important to establish whether the immotility is due to sperm degeneration and death (necrozoospermia) or existence of flagellar defects. The ultrastructural examination of the semen is strongly recommended also to prospectively evaluate the quality of spermatozoa to be utilized for extracorporeal reproductive procedures such a s in vitro fertilization and embryo transfer (IVFI ET), gamete intra-fallopian tube transfer (GIFT), etc., especially when previous attempts have been unsuccessful either due to lack of oocyte penetration or fertilization, or failure of the zygote to divide, o r to divide normally. Finally, the structural integrity of the spermatozoon should be evaluated prior to the performance of high risk medically assisted conceptional procedures such a s zona pellucida drilling and sperm micro-injection in the oocyte. With only a few exceptions, i t is difficult to think of situations in which electron microscopy plays a more important diagnostic role than that played in assessing the structural quality and functional preparedness of a population of spermatozoa for the reproductive process. This is not surprising if one

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