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

Cilia and flagella appeared very early in evolution to provide unicellular organisms with motility in water. Adaptation to non-aquatic life in plants resulted in the almost complete elimination of these organelles, except for gametic transport in some phylogenetic groups. In contrast, cilia and flagella were retained and employed for a wide variety of functions requiring fluid movement in complex multicellular animals. The functions of cilia in diverse processes such as left-right axis pattern formation, cerebrospinal fluid flow, sensory reception, mucociliary clearance and renal physiology indicate that cilia have been adapted as versatile tools for many biological processes. In this review, we discuss recent discoveries that have extended knowledge of the roles of cilia in normal development, and the pathological consequences caused by their dysfunction in mammals. We also consider evolutionary relationships between cilia from lower and higher eukaryotes, outline the ciliary components required for assembly and motility, and review the terminology of axonemal heavy chain dynein genes.

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To beat or not to beat: roles of cilia in development and disease

This review summarizes the current scientific literature concerning the ependymal lining of the cerebral ventricles of the brain with an emphasis on selective barrier function and protective roles for the common ependymal cell. Topics covered include the development, morphology, protein and enzyme expression including reactive changes, and pathology. Some cells lining the neural tube are committed at an early stage to becoming ependymal cells. They serve a secretory function and perhaps act as a cellular/axonal guidance system, particularly during fetal development. In the mature mammalian brain ependymal cells possess the structural and enzymatic characteristics necessary for scavenging and detoxifying a wide variety of substances in the CSF, thus forming a metabolic barrier at the brain-CSF interface.

The ependyma: A protective barrier between brain and cerebrospinal fluid

A balanced view of the cerebrospinal fluid composition and functions: Focus on adult humans

Eukaryotic cilia and flagella are evolutionarily conserved organelles that protrude from the cell surface. The unique location and properties of cilia allow them to function in vital processes such as motility and signaling. Ciliary assembly and maintenance rely on intraflagellar transport (IFT), the bidirectional movement of a multicomponent transport system between the ciliary base and tip. Since its initial discovery more than two decades ago, considerable effort has been invested in dissecting the molecular mechanisms of IFT in a variety of model organisms. Importantly, IFT was shown to be essential for mammalian development, and defects in this process cause a number of human pathologies known as ciliopathies. Here, we review current knowledge of IFT with a particular emphasis on the IFT machinery and specific mechanisms of ciliary cargo recognition and transport.

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The Intraflagellar Transport Machinery

Air-dried smears of saline suspensions of mammalian spermatozoa were stained for 10-60 min at room temperature in a mixture of eosin Y, fast green FCF, and naphthol yellow S (0.1% w/v, each dye) in 1.0% aqueous acetic acid. They were then blotted, rinsed in 1.0% acetic acid until no more dye was removed (0.5-1.5 min), blotted and allowed to dry completely, rinsed in xylene and mounted in synthetic resin. In all species examined except the rat, acrosomes were stained greenish blue to bluish green or blue depending on their thickness; in the rat, they displayed more affinity for eosin and were reddish. In all species, spermatozoan nuclei were strongly stained by naphthol yellow. In intact sperm heads, postnuclear cap had a yellowish green appearance. Midpieces of rodent spermatozoa, especially those of younger gametes, were stained bright red while those of ejaculated bull and rabbit spermatozoa were stained blue-green. Cytoplasmic droplets associated with rodent spermatozoa were consistently stained a dark...

An eosin-fast green-naphthol yellow mixture for differential staining of cytologic components in mammalian spermatozoa.

In the mouse seminiferous epithelium four sites of formed microtubules (MTS) are axonemes and manchettes of spermatids, Sertoli-cell cytoplasm, and spindles of dividing cells. Various experimental treatments indicate that the manchette MTS are different from other formed MTS. Fixation in 1% osmium tetroxide yields good preservation of axonemal MTS, varying degrees of preservation of manchette MTS, but no preservation of spindle or Sertoli-cell MTS. Cell-suspension, and intact-tubule cultures subjected to 0–4°C or 50°C exhibit a breakdown of spindle and Sertoli-cell MTS while manchette and axonemal MTS remain intact. Cell suspensions exposed to Colcemid or colchicine show no MTS in Sertoli and dividing cells; axonemal MTS are unaffected while in the manchette there is an increase in cross-bridging between MTS. Cell suspensions treated for 120 minutes with vinblastine sulfate do not show MTS in the Sertoli cells, but crystals presumed to be microtubule protein are present after 15 minutes of exposure. Spindle MTS are dismantled but vinblastine-induced crystals are not present even after 120 minutes of exposure. The manchette MTS are dismantled by vinblastine in a proximal direction from their distal limits. This is accompanied by the formation of crystals of microtubule protein in the spermatid cytoplasm, while axonemal MTS are unaffected. Vinblastine and Colcemid used in combination cause the disruption of Sertoli-cell MTS after 15 minutes, spindle MTS after 30 minutes and formation of crystals after 60 minutes of exposure. The manchette MTS are disassembled, the pattern is disrupted and increased numbers of cross-bridges are seen. These findings indicate that the manchette MTS might be intermediate-type microtubules since they exhibit the reactions of both cytoplasmic and flagellar-type MTS.

Ultrastructural characterization of the manchette microtubules in the seminiferous epithelium of the mouse

Gametogenesis is normal through meiosis and the earliest phases of spermiogenesis in male-sterile mice homozygous for the recessive, pleiotropic, mutation hpy (hydrocephalic-polydactyl). However, structurally complete sperm flagella were not encountered. Instead partially assembled axonemal structures and/or poorly organized aggregates of other tail components (mitochondria, outer coarse fibers) were seen at the posterior poles of nuclei in older spermatids. The ultrastructure of centrioles in spermatids was normal, but that of axonemes associated with them was not. These findings suggest that the observed flagella dysgenesis results from defects in assembly rather than from defective intiation centers. Released "gameters" usually consisted of ditorted nuclei and associated acrosome enclosed in a relatively close fitting plasma membrane. Perturbations of sperm head development were also encountered; they included extreme nuclear elongation, and distortion of the acrosome and underlying nuclear material by impushings of finger-like processes of Sertoli cells. It is believed that sperm head anomalies are secondary consequences of the mutant condition. The findings support the view that the hpy locus represents one of a number of genes primarily involved in the mediation of flagella development.

Spermatogenesis revisited. IV. Abnormal spermiogenesis in mice homozygous for another male-sterility-inducing mutation, hpy (hydrocephalic-polydactyl).

When homozygous the recessive, pleiotropic, mutation hpy (hydrocephalic-polydactyl) produces post-natal hydrocephalus, complete sterility in males, and reduced reproductive performance in females. Because the fertility problems and the development of hydrocephalus could arise as consequences of defective flagella and ciliary axonemes, this mutant type might serve as a useful animal model for the immotile cilia syndrome. Ultrastructural defects seen in axonemes of flagella, and of cilia from the trachea, oviduct, and ependyma included: a deficiency of inner dynein arms (the most frequent defect); an absence of one or both central-pair tubules; extra central tubules; a displacement of one outer doublet and/or the central-pair tubules. Some axonemes showed more than one of these defects. The frequency of dynein-deficient axonemes in all three tissues was similar (about 35%) and fell within the range reported for human patients with the immotile cilia syndrome. On this basis, this mutant type might be considered as a useful animal model for such studies. There were no indications of situs inversus, nor was there a marked increase in respiratory problems. So hpy/hpy mice do not exhibit all of the clinical symptoms characteristic of the human condition.

The immotile cilia syndrome. Mice versus man.

Sterile male mice homozygous for the recessive p-locus allende p were used. Wild-type and mutant littermates were sacrificed at 10 spaced intervals over a 6-20 week period after birth. Tissues were removed and prepared for light and electron microscopy. A minimum of 1000 cells for at least 3 animals of each genotype were observed. The course of spermatogenesis appeared to be normal until the completion of meiosis. During the Golgi phase ultrastructural appearances differed. In mutants spermatids with normal appearing ultrastructure were seen but the Golgi apparatus frequently appeared less well organized and sometimes more than 1 proacrosome was present or more than 1 specialized site. Other defects were also seen in older spermatids. Head morphology often showed blebbing and many were highly bizarre. Some spermatozoa were undergoing degeneration and phagocytosis. Flagella development and ultrastructure were normal. Dissociated axonemes and midpieces were common as were flagella without heads. Multinucleated spermatids were frequently present. Sperm motility was high. Headless spermatozoa were most active. Photographic reproductions illustrate the electron microscopic findings. Since flagella did not show any ultrastructural defects it is concluded that the action of the p mutant allele was confined to the Golgi-nucelus complex. Therefore the development of the sperm head is probably under a group of genes different from those of the flagella.

John H. D. Bryan

Papers

An eosin-fast green-naphthol yellow mixture for differential staining of cytologic components in mammalian spermatozoa.

Ultrastructural characterization of the manchette microtubules in the seminiferous epithelium of the mouse

Spermatogenesis revisited. IV. Abnormal spermiogenesis in mice homozygous for another male-sterility-inducing mutation, hpy (hydrocephalic-polydactyl).

The immotile cilia syndrome. Mice versus man.

Spermatogenesis revisited. III. The course of spermatogenesis in a male-sterile pink-eyed mutant type in the mouse.