Spermatogenesis in Caenorhabditis elegans, as in most animals, is a differentiation pathway in which spermatogonial stem cells differentiate into spermatozoa. This process involves mitotic proliferation of spermatogonial cells to form primary spermatocytes and two subsequent meiotic divisions of the nucleus during spermatid formation. Spermiogenesis then follows, which is the maturation of spermatids into spermatozoa. As in other nematodes, C. elegans spermatozoa lack an acrosome and flagellum (for review, see Foor 1983) and move by crawling across the substrate (for review, see Theriot 1996). Although C. elegans sperm differ from flagellated sperm in a number of significant ways, both types of sperm engage in meiosis and in unusual cell divisions characterized by extremely asymmetric cytoplasmic partitioning.
C. elegans offers several advantages over other organisms in which spermatogenesis has been studied. Primary spermatocytes differentiate into spermatids in only 90 minutes and wild-type cells can be easily studied under simple culture conditions in vitro (Ward et al. 1981; L'Hernault and Roberts 1995). Differentiation of spermatocytes into spermatids in vivo is much slower in organisms that produce flagellated sperm, taking about 32 days in humans, 24 days in rats (for review, see Fawcett 1994), and 5 days in Drosophila (Lindsley and Tokuyasu 1980). Although in vitro development of mouse sperm is possible, it has only been attained when germ cells are cocultured with transformed Sertoli-like cells under complex culture conditions (Rassoulzadegan et al. 1993; Hofmann et al. 1994, 1995). Most animals produce sperm that can only be studied by microscopic techniques following dissection of the testes. In contrast, C. elegans is small and transparent, permitting microscopic observations of sperm development and motility in a living, undissected animal (Ward and Carrel 1979). Finally, C. elegans sperm develop without any of the accessory cells that complicate analysis of flagellated sperm development (for review, see Skinner et al. 1991). The steps of C. elegans spermatogenesis have been determined by light and electron microscopy (Klass et al. 1976; Wolf et al. 1978; Ward et al. 1981), and they have also been studied in a large collection of mutants (Fig. 1).
In hermaphrodites, spermatogenesis begins during the L4 larval stage and is completed in the young adult shortly after molting, requiring about 6–7 hours at 25°C (Hirsh et al. 1976). In males, spermatogenesis starts in the L4 and continues through adult life. Although the gonad anatomies of hermaphrodites and males differ (see Schedl; Emmons and Sternberg; both this volume), the process of spermatogenesis is similar at the cellular level. Spermatozoa derived from males closely resemble those of the hermaphrodite, with a few exceptions noted below.
Spermatocytes initially form in a syncytium with a central cytoplasmic core named the rachis (Fig. 1A) (Hirsh et al. 1976). The stages of cellular differentiation within the germ line proceed from the distal tip toward the seminal vesicle (in males; see Emmons and Sternberg, this volume) or the spermatheca (in hermaphrodites; see Schedl, this volume). Mitotic proliferation of the spermatogonial nuclei occurs near the distal tip. As the syncytial nuclei approach the loop region, where the gonad turns posteriorly, meiosis is initiated and a zone of nuclei in pachytene of the first meiotic division can be identified (see Schedl, this volume). At this point, spermatocytes bud off the rachis (Fig. 1B), and individual cells complete meiosis. There is no apparent analog of the Sertoli cell, which is the somatic cell component of the mammalian seminiferous epithelium that provides nourishment and structural support to developing sperm cells (for review, see Skinner et al. 1991). C. elegans spermatocytes completely separate as each one buds from the rachis, while adjacent mammalian spermatocytes form a syncytium (for review, see Fawcett 1994). Metaphase I follows shortly with either of two results (Fig. 1) (Ward et al. 1981): The primary spermatocyte can divide completely into two secondary spermatocytes (Fig. 1D) or it can incompletely divide, with the two halves remaining in syncytium (Fig. 1D′). In both cases, the second meiotic division occurs with concomitant formation of a cytoplast, called the residual body, between the developing spermatids (speckled structures at D and D′ in Fig. 1). In many mammalian species, a residual body does not form until after release of elongate spermatids, well after completion of meiosis (for review, see Fawcett 1994). Mammalian spermatogenesis is characterized by a lengthy period of differentiation between completion of meiosis and conversion of the spermatid into the spermatozoon. After a C. elegans spermatid has budded from the residual body, it is not known whether there are required differentiation steps that must occur prior to competence to initiate spermiogenesis.
Spermiogenesis occurs in hermaphrodites when spermatids are moved into the spermatheca. In males, spermatids are stored in the seminal vesicle until ejaculation into the hermaphrodite. Shortly after ejaculation, spermatids (Figs. 1E and 2a) differentiate into spermatozoa (Figs. 1H and 2b,c). This differentiation occurs within the uterus just inside the vulva, and some spermatids that remain in the male after copulation also undergo spermiogenesis (Ward and Carrel 1979). C. elegans spermatozoa, like all examined nematode spermatozoa (Foor 1983), lack both a flagellum and an acrosome (Wolf et al. 1978; Ward et al. 1981). Instead, the C. elegans spermatozoan cell body has a single pseudopod (Fig. 2), and directed membrane flow allows it to crawl over the substratum (Figs. 1H and 2b) (Nelson et al. 1982; Roberts and Ward 1982a,b). All mitochondria, the nucleus, and other organelles reside only in the cell body, which is separated from the pseudopod by laminar membranes (Fig. 2c). Much of the in vivo development discussed above can also be studied in vitro because spermatocytes that have detached from the rachis can complete differentiation into spermatozoa in simple media (for review, see L'Hernault and Roberts 1995; K. Machaca and S. L'Hernault, unpubl.).
The unusual reproductive biology of C. elegans offers advantages for mutational analyses of spermatogenesis. Hermaphrodite self-fertilization is extraordinarily efficient, and nearly every spermatozoon successfully fertilizes an oocyte in the young hermaphrodite (Ward and Carrel 1979). Whereas young wild-type hermaphrodites usually lay eggs containing developing embryos, mutant self-sterile hermaphrodites that contain defective sperm lay oocytes. When such mutants are mated to wild-type males, cross-progeny form, demonstrating that normal spermatozoa can rescue the sterile phenotype of the mutant hermaphrodite. A genetic screen using this strategy has identified more than 60 genes that affect spermatogenesis (Hirsh and Vanderslice 1976; Ward and Miwa 1978; Argon and Ward 1980; Ward et al. 1981, 1982, 1983; Edgar 1982; Nelson et al. 1982; L'Hernault et al. 1987, 1988, 1993; Shakes 1988; Shakes and Ward 1989a,b; L'Hernault and Arduengo 1992; Varkey et al. 1993, 1995; Minniti et al. 1996). Figure 1 summarizes how some of these mutants alter or arrest spermatogenesis. Many of these mutants appear to be spermatogenesis-specific in their mutant defects (see, e.g., L'Hernault et al. 1988), and, in those cases that have been analyzed, transcription of the encoded gene product is limited to the male germ line (L'Hernault and Arduengo 1992; L'Hernault et al. 1993; Varkey et al. 1995; Minniti et al. 1996). The phenotypes of many of these mutants are discussed below in the context of normal sperm function. Table 1 summarizes all of the mutants discussed in this chapter.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor (NY)
Riddle DL, Blumenthal T, Meyer BJ, et al., editors. C. elegans II. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 1997. Section I, Overview.