The genetics and cell biology of spermatogenesis in the nematode C. elegans

Molecular and Cellular Endocrinology 306 (2009) 59–65

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Review

The genetics and cell biology of spermatogenesis in the nematode C. elegans Steven W. L’Hernault ∗ Department of Biology, Emory University, 1510 Clifton Road NE, Atlanta, GA 30322, USA

a r t i c l e

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Article history: Received 11 August 2008 Received in revised form 6 January 2009 Accepted 8 January 2009 Keywords: Spermatogenesis Fertilization Meiosis Reproduction C. elegans

a b s t r a c t Creation of mutants that affect spermatogenesis is very challenging in most experimental systems, especially mammals. The main reason this is true is because “absence of successful mating” is a negative result that can occur for a wide variety of trivial, irrelevant reasons. The C. elegans hermaphroditic mode of reproduction has unusual features that facilitate analysis of spermatogenesis. Normally, hermaphrodites are virtually 100% self-fertile and spermatogenesis defective mutants are self-sterile. A candidate spermatogenesis defective mutant will produce cross-progeny after mating to a wild type male, showing that the presence of sperm is both necessary and sufficient to restore fertility to the sterile hermaphrodite. This has allowed selection of a large number of spermatogenesis defective mutants. In this article, I will review spermatogenesis, how mutants are made and what has been learned about the identified genes and their roles during development and fertilization. © 2009 Elsevier Ireland Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Overview of C. elegans reproductive biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Physiology of spermatogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cytology of C. elegans spermatogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mutants that affect spermatogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mutants that affect sperm meiosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mutants affecting FB–MOs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mutants that affect the cytoskeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sex-specific aspects of spermiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fertilization defective mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Spermatogenesis mutants that act within the embryo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Overview of C. elegans reproductive biology Dioecious species require mating in order for propagation to occur, and mating requires that males be able to chase and inseminate females. C. elegans has conventional males but has a hermaphrodite instead of a standard female. Similar to dioecious organisms, a C. elegans male can mate with a hermaphrodite and produce cross-progeny. However, under typical laboratory circumstances, the hermaphrodite is self-fertile and no contact between a male and female is required for species propagation (Brenner, 1974). As described below, this feature greatly facilitates

∗ Tel.: +1 404 727 4204; fax: +1 404 727 2880. E-mail address: [email protected]. 0303-7207/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2009.01.008

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the recovery and analysis of mutants in which spermatogenesis is defective. The C. elegans embryonic hermaphrodite forms two primordial germ cells that begin mitotic proliferation after hatching. The gonad epithelium is a syncitium where nuclei share a common cytoplasm within a hollow tube, and there are two gonad arms that are mirror image symmetrical about a single vulval opening to the exterior. The first phase of germ cell proliferation ends when 45–75 (inversely correlated with the growth temperature) spermatogonia exit mitosis, commit to meiosis, bud from the central, acellular cytoplasmic core named the rachis (Fig. 1A) and differentiate into 180–300 haploid spermatids. The gonad then switches identity and begins oogenesis as the L4 stage hermaphrodite molts into an adult (Hirsh et al., 1976). The first ovulation pushes the spermatids out of the gonad and into the spermatheca, where spermatids rapidly

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Fig. 1. (A) Summary of spermatogenesis. Asymmetric partitioning of cellular constituents occurs at three points during C. elegans spermatogenesis, as indicated by doubleheaded red arrows at the red numbers. 1, syncytial pachytene spermatocytes with many FB–MOs bud from the rachis and divide to form secondary spermatocytes (FB–MOs are shown in green); 2, spermatids selectively retain FB–MOs as they bud from the residual body; 3, MOs fuse with the spermatid plasma membrane as a pseudopod extends from the cell body during spermiogenesis. Nuclei are the circles in the center of each cell. Nuclei are patterned with lines to represent stages when chromatin is in condensed meiotic chromosomes or filled (in black) after chromatin forms a single highly condensed sphere. (B) Summary of morphogenesis of the FB–MO complex. 1, the fibrous body develops in close association with, and is surrounded by, the membranous organelle (MO) within the primary spermatocyte. The MO is separated by a collar region (c) into a head (h; speckled region at left) and body (b; region to the right of the collar); 2, the FB–MO complex reaches its largest size within primary spermatocytes. The double-layered MO-derived membrane surrounds the FB, which is composed of the major sperm protein filaments; 3, the MO-derived membranes surrounding the FB retract and fold up while FB filaments depolymerize and disperse as spermatids bud from the residual body; 4, the head of each MO (arrow) moves to a position just below the plasma membrane (pm) of the spermatid after the FBs have depolymerized and disappeared. The irregular shapes within the FB represent retracted membrane that had covered the exterior of the FB; 5, the head of the MO fuses at the collar to the plasma membrane and exocytoses its contents (dots at arrow) onto the cell surface. A permanent fusion pore remains at the point of each MO fusion (each cell has many MOs; B is modified from Arduengo et al. (1998). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

differentiate into spermatozoa. These spermatozoa spend the rest of their life waiting for oocytes to be ovulated into the spermatheca, which is the location where fertilization occurs (Ward and Carrel, 1979). Such a fertilized oocyte completes meiosis, forms a chitin eggshell and transitions into an embryo as it exits the spermatheca into the uterus. Embryos in the uterus move away from the spermatheca and are pushed out into the environment through the vulva, about 3.5 h after conception (Hirsh et al., 1976). Spermatogenesis in males is similar to that which occurs in hermaphrodites (Klass et al., 1976). Unlike hermaphrodites, the male gonad is a single reflexed structure and it is connected to the copulatory spicules located in the male tail region. Like hermaphrodites, spermatogenesis begins during the L4 larval stage, but it continues throughout the adult stage. Spermatids are stored in the seminal vesicle and become spermatozoa during ejaculation into a hermaphrodite. Spermatids become spermatozoa when they are exposed to a factor in seminal fluid during ejaculation (Ward and Carrel, 1979). Male-derived spermatozoa are ∼50% larger than those produced by the hermaphrodite, likely due to selection on male sperm to outcompete hermaphrodite sperm (LaMunyon and Ward, 1995, 1998). It is possible to purify large numbers of sperm from males that are suitable for biochemistry (reviewed by L’Hernault and Roberts, 1995). 2. Physiology of spermatogenesis Once nuclei within the germ cell syncitium exit mitosis and commit to meiosis, 4N spermatocytes become individualized cells separated from each other by a plasma membrane (Fig. 1A) (Wolf

et al., 1978; Ward et al., 1981). Cellularized spermatocytes are able to complete meiosis and differentiate into spermatozoa in vitro in a simple, chemically defined medium where the most complex organic compound is glucose (Nelson and Ward, 1980; Machaca et al., 1996). Spermatocytes can complete differentiation into spermatids in the absence of any type of accessory cell or added hormone in vitro and, presumably, in vivo. In hermaphrodites, the spermatid–spermatozoon transition probably requires a paracrine factor in vivo. Spermatids are presumably exposed to this currently unknown factor as they are pushed into the spermatheca during the first ovulation (Ward and Carrel, 1979). C. elegans hermaphrodites are extraordinarily efficient at reproduction. In a young, wild type hermaphrodites, a spermatozoon fertilizes each oocyte as it enters the spermatheca. Anatomically, the spermatheca is only slightly larger than the diameter of the oocyte and it has a myofibrillar layer, which contracts to expel the fertilized oocyte. Expulsion of the fertilized oocyte displaces many spermatozoa out of the spermatheca into the uterus. Fertilization is not known to occur in the uterus, and such displaced spermatozoa rapidly move back into the spermatheca (Ward and Carrel, 1979). This process of displacement can occur hundreds of times as each spermatozoon competes to fertilize an oocyte. Eventually, each sperm fertilizes an oocyte and the hermaphrodite continues to produce ∼40–100 oocytes that are subsequently pushed out into the environment. At this point, the hermaphrodite stops ovulating. However, if the hermaphrodite is inseminated by a male, ovulation resumes and cross-progeny will form. Such an inseminated hermaphrodite can produce many additional progeny (Ward and Carrel, 1979; Hodgkin, 1983). This indicates that hermaphrodites

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commit resources to ovulation when sperm are present, and a signaling system between sperm and oocytes is known to facilitate this process (reviewed by Greenstein, 2005).

3. Cytology of C. elegans spermatogenesis Detailed cytological analysis of spermatogenesis in vitro has been performed (Fig. 1A). Primary spermatocytes undergo meiosis I to yield two secondary spermatocytes that can divide into two distinct cells (∼60% of the time; not shown) or remain attached (∼40% of the time; Fig. 1A2). Each secondary spermatocyte undergoes an asymmetric division to yield two haploid spermatids (Ward et al., 1981). Spermatids bud from an acellular residual body (Fig. 1A2) into which are placed components not required for sperm function. These components include all ribosomes, nearly all actin and myosin and all of the tubulin except for what remains in the single centriole. The mature spermatid retains the highly condensed haploid nucleus, the centriole embedded in a RNA rich perinuclear layer that surrounds the nucleus, the mitochondria and unusual Golgiderived secretory membranous organelles (MOs) (Ward, 1986). The transition of spermatids into spermatozoa (Fig. 1A3) occurs in the natural absence of protein synthesis, since all ribosomes were discarded into the residual body during spermatid budding (Ward, 1986). Like other nematodes, C. elegans spermatid activation causes the cell to extend a single pseudopod; there is no flagellum (Wolf et al., 1978; Nelson and Ward, 1980). This pseudopod is not like that found in other amoeboid cells, such as white blood cells or free-living amoeba because, once formed, its position remains fixed relative to the cell body. Another difference is that nematode sperm do not use actin for motility but, rather, uses a filamentous network composed of major sperm protein (MSP) (Ward and Klass, 1982; Roberts and Stewart, 1997). Despite these differences from other amoeboid cells, the simplicity of this experimental system has facilitated detailed cellular and molecular analyses, mostly in the parasitic nematode Ascaris, where the availability of large amounts of material has facilitated biochemical analyses (reviewed by Stewart and Roberts, 2005). The MSP used for pseudopodial motility is delivered to spermatids in association with the MOs (Wolf et al., 1978; Ward et al., 1981; Ward and Klass, 1982). MOs play a role that in some ways is analogous to that played by the acrosome in flagellated sperm. The MOs first appear at the periphery of the Golgi apparatus in syncitial pachytene spermatocytes (Wolf et al., 1978; Roberts et al., 1986). As MOs form, a fibrous body (FB) composed of bundled major sperm protein filaments forms in association with each one (Fig. 1B1). A double-layered membrane derived from the MO envelopes each FB, and FB–MOs reach their maximum size in secondary spermatocytes (Fig. 1B2). The double-layered membrane is retracted from around the FB as spermatids bud from the residual body (Fig. 1B3). The exposed FBs depolymerize and MSP monomers disperse throughout the cytoplasm of the budded spermatid (Ward and Klass, 1982). As spermatids (Fig. 1B4) activate into spermatozoa (Fig. 1B5), the MSP monomers polymerize to form a filamentous network in the pseudopod (Roberts, 1983), which was subsequently shown to mediate cell motility in the nematode Ascaris (Sepsenwol et al., 1989). The MO functions as a secretory vesicle, permanently docking with the cell surface (Wolf et al., 1978), where it deposits transmembrane proteins (Xu and Sternberg, 2003; Chatterjee et al., 2005) and glycoproteins (Roberts and Ward, 1982). As discussed below, many spermatogenesis mutants affect MO morphogenesis or function. Spermatid activation (spermiogenesis) has been studied in vitro and a variety of treatments stimulate this cellular transition. The cationic ionophore monensin was the first treatment shown to cause spermiogenesis (Nelson and Ward, 1980). Monensin elevates

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the intracellular pH by ∼0.5 pH unit and weak unprotonated bases, such as triethanolamine, can also cause spermiogenesis. Protease treatment of the cell surface will also cause spermiogenesis, and either trypsin or Pronase will work (Ward et al., 1983). Trifluorperazine, chlorpromazine and W7 cause wild type spermatids to extend spikes after treatment. Washing the drug out of the medium causes the spikes to disappear and a single pseudopod to form. While these drugs are all known to antagonize calmodulin function (Van Belle, 1984), it is unclear if this is the mechanism by which they operate in this system (Shakes and Ward, 1989a). Patch clamp electrophysiological experiments revealed that inhibiting a voltage gated inwardly rectified Cl− channel with the anion channel inhibitor DIDS was correlated with spermatid activation. How, or if, ion channel inhibition is related to spermatid activation remains unclear (Machaca et al., 1996). It is not yet possible to use C. elegans in vitro fertilization to examine spermatozoa–oocyte interactions. However, injecting the contents of a sperm-loaded micropipet through the vulva allows artificial insemination. This technique revealed that spermatozoa formed by Pronase treatment are not fertilization-competent while triethanolamine activated spermatids can fertilize oocytes that become normal progeny (LaMunyon and Ward, 1994). 4. Mutants that affect spermatogenesis C. elegans hermaphrodite self-reproduction has several significant advantages for the recovery and maintenance of spermatogenesis defective mutations. Normally, young hermaphrodites fertilize virtually all ovulated oocytes and lay self-progeny (Ward and Carrel, 1979). After random mutagenesis, individual F2 mutant hermaphrodites that lay unfertilized oocytes onto their growth plate are easily identified. Such self-sterile hermaphrodites can be placed with wild type males and allowed to mate. If they produce cross-progeny, this means that wild type sperm are both necessary and sufficient to correct the self-sterile phenotype of the mutant hermaphrodite (Argon and Ward, 1980; L’Hernault et al., 1988). This means that spermatogenesis defective (spe or fer) mutants can be identified under circumstances (in hermaphrodites) that facilitate their recovery (some relevant mutants cause a Spe phenotype, but have other names). Furthermore, no special forethought or strategy is required to ensure the mutation will be maintained in the F3 and later generations. There are two ways of determining the number of genes that are sperm-specific in their effect. Microarray experiments reveal that greater than 400 genes show expression that is up-regulated during spermatogenesis (Reinke et al., 2000). The second way is to create mutants that specifically affect spermatogenesis. So far, 44–60 spe genes have been identified for which at least one mutant allele has been recovered. Statistical analysis of the frequency distribution of mutations suggests that there are about ∼90 genes for which the loss of function yields a Spe phenotype (L’Hernault et al., 1988; S.W. L’Hernault, unpublished). Consequently, about twothirds of the C. elegans genes predicted to have a loss of function Spe phenotype are already identified as mutants (S.W. L’Hernault, unpublished data). Perhaps many genes that show up-regulated expression in microarray experiments are redundant with other genes and null mutations in them will prove to have either no or a subtle phenotype. Currently, the DNA sequences of 28 genes known to affect spermatogenesis have been determined, and many of these are discussed below and shown in Fig. 2. 5. Mutants that affect sperm meiosis Wee1P type kinases were first identified in fission yeast (reviewed by Nurse, 2000) and, like other animals, C. elegans

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Fig. 2. (A) Stages of wild type spermatogenesis are shown diagramatically as an ordered pathway of morphogenesis with the stages labeled in blue. Some of the >44 known genes are placed on the pathway where ultrastructural or light microscopic defects are evident. The ife-1 gene is not shown, but it slows down the rate of spermatogenesis and also affects spermatozoa. The next to last step of the pathway represents mutant spermatozoa that are cytologically normal but cannot enter oocytes. The spe-11 mutant is a paternal effect lethal mutant that makes spermatozoa that enter the egg and the resulting defective embryo dies. (B) The most common terminal stages of mutants that arrest morphogenesis without forming spermatids; (C) the protein distribution of SPE-9 (arrows), the hypothesized SPE-9 receptor (“Y” on egg surface), the SPE-38 protein (green arrows) and the SPE-41/TRP-3 protein (red dots). Please note that individual drawings are not to scale; for instance, spermatozoa are 5–6 mm in diameter while the eggs shown are ∼45 mm (modified from L’Hernault, 2006). (These two figures and their legends first appeared in L’Hernault (2006) Copyright: © 2006 S.W. L’Hernault, distributed under the terms of the Creative Commons Attribution. This figure has been modified from the original version to reflect scientific advances.) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

has a homolog that is widely expressed (Burrows et al., 2006; Lamitina and L’Hernault, 2002). Nonetheless, six dominant wee1.3(gf) mutants that display a Spe phenotype, with defects confined to spermatogenesis, were identified. WEE-1.3 most likely acts by negatively regulating ncc-1 (Boxem et al., 1999), which is the cdc2p ortholog that regulates both mitotic and meiotic divisions in C. elegans, as it does in other eukaryotes (Lundgren et al., 1991). Null wee-1.3 mutants have an embryonic/larval lethal phenotype, which is not surprising since this gene likely regulates all cell divisions (Lamitina and L’Hernault, 2002). Each dominant Spe wee1.3 missense mutation changes a base near the C-terminus. These dominant Spe wee-1.3 mutants arrest at the primary spermatocyte stage with an intact nuclear envelope and a nucleus that arrests at pachytene of meiosis I. This 4N nucleus has highly condensed chromatin with ultrastructure similar to that of the 1N spermatid nucleus (Fig. 2B). These mutant primary spermatocytes do not divide and their undivided nucleus is usually opposite vacuolated FB–MOs. These dominant wee-1.3 mutants reveal that the G2/M transition of male meiosis I must utilize a unique pathway (Lamitina and L’Hernault, 2002). C. elegans controls RNA stability and translation with pumiliolike RNA binding proteins encoded by puf genes. PUF-8 is required for primary spermatocytes to complete meiosis I prophase, and when it is absent, meiosis stops and cells re-enter mitosis and become tumorous germ cells (Fig. 2A). This indicates that primary spermatocytes require PUF-8 to complete meiosis and spermatogenesis (Subramaniam and Seydoux, 2003). 6. Mutants affecting FB–MOs spe-39 mutants arrest as aberrant spermatocytes that lack ∼800 nm MOs but, rather, have many ∼100 nm vesicles that are probably MO intermediates (Fig. 2B). FBs are present but they are variable in shape and not surrounded by membranes. SPE-39 is a novel protein that is diffusely distributed in sperm and many other C. elegans cell types, and RNAi of this gene causes embryonic lethality. Every animal species investigated has a spe-39 ortholog, but

yeast and other unicellular organisms lack an obvious homolog (Zhu and L’Hernault, 2003). Recent data indicates that SPE-39 is a new component of the vesicular trafficking pathway in animals that plays a role in cargo delivery to lysosomes. This suggests that the MO secretory vesicles in C. elegans sperm are specialized lysosomes, consistent with other observations (Zhu et al., 2009). spe-6 null mutants do not form FB–MOs because they cannot make FBs from MSP, and they arrest development as terminal spermatocytes (Fig. 2B) (Varkey et al., 1993). SPE-6 is a casein I type serine threonine kinase (Muhlrad and Ward, 2002), suggesting that phosphorylation regulates FB formation in an as yet unknown way. Certain spe-6 hypomorphic mutants are bypass suppressors of the spe-8 pathway (see below) and these mutants form normalappearing FB–MOs and some spermatids. spe-4 null mutants arrest spermatogenesis as terminal spermatocytes (Fig. 2B), and they contain FBs that are not associated with its abnormally vacuolated MOs. SPE-4 is a presenilin, which are aspartyl proteases that cleave membrane proteins. Human presenilin mutants incorrectly digest the Alzheimer precursor protein (Xia and Wolfe, 2003) and show early-onset disease (Rogaev et al., 1995). SPE-4 protein localizes within FB–MOs but its substrate(s) has/have not yet been identified (Arduengo et al., 1998). A recently published paper (Gosney et al., 2008) reports a nonnull spe-4 mutant that suppresses mutants in the spe-8 pathway (see below). spe-5 null mutants usually form terminal spermatocytes that have four haploid nuclei and do not form spermatids (Fig. 2B). Occasionally, spe-5 mutant hermaphrodites produce a few self-progeny, so a few spermatozoa must be present (Machaca and L’Hernault, 1997). The FB–MOs that form in spe-5 mutants are vacuolated but, unlike spe-4 mutants (see above), each is still associated with a FB. SPE-5 is a vacuolar ATPase B subunit (P. Hartley and S.W. L’Hernault, unpublished) that is found associated with the FB–MOs (E.J. Gleason and S.W. L’Hernault, unpublished). The V-ATPase is a highly conserved complex of at least 13 polypeptides that hydrolyzes ATP to pump protons across biological membranes (Nishi and Forgac, 2002). This suggests that the ultrastructural defects observed in

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spe-5 FB–MOs are due to defects in acidification of this compartment. spe-17 mutants form abnormally small spermatids (∼66% the size of wild type) that retain ribosomes as these cells bud from the residual body (Fig. 2). These retained ribosomes stud the surface of the MO, a phenomenon that is never observed during wild type spermatogenesis. The associated FBs usually disassemble so that MSP disperses in the cytoplasm but MOs frequently do not fuse with the cell surface during spermiogenesis (Shakes and Ward, 1989b). The spe-17 gene encodes a small soluble protein with no obvious conserved domains or homologs outside nematodes (L’Hernault et al., 1993). Despite these abnormalities, some motile spermatozoa can still form in spe-17 mutants, which have a brood that is ∼10% the size of wild type. Like spe-17, spe-10 mutants make errors during spermatid budding from the residual body and they form spermatids that are ∼66% the size of wild type (Fig. 2). This mutant forms cytologically normal FB–MOs, but many FBs are left in the residual body during spermatid budding. The MOs in spe-10 spermatids become vacuolated and do not fuse with the plasma membrane. Furthermore, since these spermatids lack most of their MSP, they are immotile (Shakes and Ward, 1989a). SPE-10 is a four-pass integral membrane protein that is a predicted palmitoyl transferase containing a DHHC domain. This suggests that SPE-10 regulates FB–MO function by posttranslational palmitoylation of what is currently an unknown protein or proteins (Gleason et al., 2006). fer-1 mutants complete the early stages of spermatogenesis and make normal-appearing spermatids. However, MOs fail to fuse with the plasma membrane, and fer-1 mutant spermatozoa have short, immotile pseudopods (Fig. 2) (Ward et al., 1981; Roberts and Ward, 1982). FER-1 is a one pass transmembrane protein with homology to seven human dysferlin genes (Achanzar and Ward, 1997), and one is mutant in limb girdle muscular dystrophy (Bashir et al., 1998). Muscle membrane repair is defective in this disease (Bansal and Campbell, 2004) and, like its vertebrate homologs, FER-1 regulates Ca2+ mediated membrane fusion (Washington and Ward, 2006). 7. Mutants that affect the cytoskeleton spe-26 mutants form terminal spermatocytes where meiosis forms four haploid nuclei or up to 12 DAPI positive DNA masses (Fig. 2B) (Varkey et al., 1995). Sometimes, spermatids are created and these occasionally become spermatozoa, but these spermatozoa do not fertilize oocytes in null spe-26 mutants. The spe-26 gene encodes a kelch homolog so it presumably functions as an actin binding protein that facilitates transport of organelles into developing spermatids. spe-15 mutants make errors during spermatid budding from the residual body (Fig. 2). The components that are normally placed into either the residual body or the spermatid are distributed to both compartments during mutant spermatogenesis. SPE-15 is a myosin VI, so actomyosin motility is responsible for the asymmetric partitioning of components during spermatid budding from the residual body. spe-15 mutants make spermatids but in vitro activation with protease results in spermatozoa with abnormal pseudopods (Kelleher et al., 2000). 8. Sex-specific aspects of spermiogenesis Five genes that exhibit hermaphrodite-specific spermiogenesis defects have been discovered. Each mutant produces cross-fertile males and self-sterile hermaphrodites and they form the spe8 pathway, after the first discovered member (L’Hernault et al., 1988).

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SPE-8 is a non-receptor tyrosine kinase with a predicted SH2 domain (P.J. Muhlrad and S. Ward, unpublished). Its substrates are currently unknown. SPE-12, SPE-19 and SPE-29 are all novel, single pass transmembrane proteins predicted to be in the plasma membrane (Geldziler et al., 2005; Nance et al., 1999, 2000). SPE-27 is a small (∼15 kD) hydrophilic protein with no previously described structural motifs (Minniti et al., 1996). Mutant hermaphrodites for any spe-8 pathway gene accumulate spermatids that do not become spermatozoa in vivo (Fig. 2). In contrast, mutant males for any spe-8 pathway gene produce spermatozoa that are fertilization-competent and these males sire cross-progeny after mating. The sizes of cross-broods sired by spe-8 (Nance et al., 1999), spe-19 (Geldziler et al., 2005) or spe29 mutant males (Nance et al., 2000) are close to that of wild type controls, while spe-12 males show a ∼7-fold reduction in cross-fertility relative to controls (Nance et al., 1999, 2000); spe-27 mutant males cross-fertility has not been quantitated. Treatment of hermaphrodite-derived spermatids with seminal fluid from males causes them to activate and become fertilization-competent. Unlike wild type, exposure of spermatids from either males or hermaphrodites to the in vitro activator Pronase causes them to extend spikes that do not become a pseudopod. However, mutant spermatids treated with the weak base triethanolamine activate into spermatozoa with normal-appearing pseuodpods (Shakes and Ward, 1989b). spe-27 self-sterile mutants were mutagenized and self-fertile hermaphrodites containing mutations within other genes were identified. Non-null spe-4, spe-6 and several as yet unidentified mutants were selected by this approach. Suppression is not specific for spe-27 because any one of the five spe-8 pathway mutants has elevated self-fertility when they also have a spe-6 suppressor mutation. SPE-6 kinase activity probably blocks spermiogenesis until the spe-8 pathway receives an activation signal (Muhlrad and Ward, 2002). spe-6 suppressor mutants show precocious spermiogenesis, suggesting that spermatid activation can occur when SPE-6 activity is reduced. Mutant males, but not hermaphrodites, contain many necrotic sperm and rarely sire progeny, suggesting that reduced SPE-6 activity results in sex-specific cell death (Muhlrad and Ward, 2002). Recently, a non-null allele of spe-4 was shown to restore partial self-fertility to spe-27 mutants. This suggests that one function of the SPE-4 presenilin is to regulate proteolytic processing of membrane proteins important for spermiogenesis (Gosney et al., 2008). These data suggest that spermiogenesis in males differs from that of hermaphrodites. Hermaphrodites use two kinases (SPE-6 and SPE-8), one soluble protein (SPE-27) and three transmembrane proteins (SPE-12, SPE-19 and SPE-29) to set up what appears to be a signal transduction pathway. Although the signal that causes spermiogenesis in hermaphrodites is unknown, it is probably sent as spermatids are pushed into the spermatheca during the first ovulation. Males use SPE-6, but do not require the other five genes for spermiogenesis. Recently, the swm-1 gene was recovered in a screen for malespecific spermatogenesis defects and it encodes a protein that appears to be a protease inhibitor. swm-1 mutant males accumulate many spermatozoa because normal inhibition of spermiogenesis does not occur. swm-1 males show greatly reduced cross-fertility, presumably because sticky spermatozoa clog the male reproductive tract and interfere with sperm transfer during copulation. The swm-1 mutant does not detectably affect hermaphrodite selffertility in otherwise wild type animals. However, swm-1 spe-8 (or other mutants in the spe-8 group) double mutant hermaphrodites show elevated self-fertility, indicating that swm-1 must be present in hermaphrodites. These data suggest spermiogenesis utilizes one or more proteases, but that it is only required in the male

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reproductive tract (Stanfield and Villeneuve, 2006). The identity of this protease(s) has not yet been determined.

tein that is both required for embryogenesis and normally has a sperm-specific expression pattern.

9. Fertilization defective mutants

Acknowledgements

Seven mutants complete spermatogenesis and can form spermatozoa that are defective in fertilization but otherwise normal, and the five genes that have been cloned are discussed below (Fig. 2). SPE-9 is a one pass integral membrane protein located in the plasma membrane of spermatids that becomes confined to the pseudopodial plasma membrane of spermatozoa (Fig. 2C). This protein has 10 EGF-like repeats located in the extracellular space, and such motifs frequently have adhesive and/or ligand-receptor roles (Singson et al., 1998). Deletion and point mutations in SPE-9 EGF repeats were used to reveal the ones that were most important during fertilization (Putiri et al., 2004). SPE-41/TRP-3 is a TRP calcium channel protein that functions during store- and, maybe, receptor-operated calcium influx, probably when fertilization is occurring. SPE-41/TRP-3 localizes within the spermatid MO but is placed into the plasma membrane of both the cell body and pseudopod of spermatozoa after MO fusion with the plasma membrane (Fig. 2C) (Xu and Sternberg, 2003). SPE-38 is a tetraspan integral membrane protein that plays an as yet to be determined role during fertilization. Structurally similar proteins play roles important for cell–cell interactions in a variety of cell types (Hemler, 2003). SPE-38 is found in MOs of spermatids and localizes to the pseudopodial plasma membrane when the MOs fuse with the plasma membrane during spermiogenesis (Fig. 2C) (Chatterjee et al., 2005). FER-14 is a one pass transmembrane protein that is nematode specific and has no obvious structural motifs (T. Kroft, E.J. Gleason and S.W. L’Hernault, unpublished). SPE-42 is a six pass transmembrane protein with a RING finger at its C-terminal end (Kroft et al., 2005). All multicellular animals, including C. elegans, encode two SPE-42 like homologs. One of these homologs is expressed during Drosophila spermatogenesis, where it is important for fertilization (Wilson et al., 2006). One human homolog has a splice form expressed in testes, suggesting it might play a role during spermatogenesis. All fertilization defective mutants contact oocytes in the spermatheca but do not fertilize them. When fer-14, spe-9, spe-13, spe41/trp-3 and spe-42 males are mated to wild type hermaphrodites they become sterile because defective male-derived spermatozoa out-compete endogenous hermaphrodite-derived spermatozoa for the limited space available in the spermatheca (Xu and Sternberg, 2003; Chatterjee et al., 2005; Kroft et al., 2005; Singson et al., 1999). The result is that oocytes only encounter defective spermatozoa when they are in the spermatheca and fertilization does not occur.

The author would like to thank the members of his lab for useful discussions. This material is based upon work supported by the National Science Foundation under Grant No. IOB-0544180 and the National Institutes of Health under Grant No. GM082932.

10. Spermatogenesis mutants that act within the embryo spe-11 mutant spermatozoa can fertilize oocytes, but embryogenesis never begins properly and the defective embryo always dies (L’Hernault et al., 1988). Ultrastructural examination of spe11 mutant spermatids reveals that they have defects in perinuclear material surrounding the nucleus (Hill et al., 1989), which is where the SPE-11 protein localizes in wild type spermatids (Browning and Strome, 1996). This sperm-derived material is required for meiotic cytokinesis in the post-fertilization egg (McNally and McNally, 2005). Other work has shown that the C. elegans sperm-derived nucleus (Sadler and Shakes, 2000), centriole (O’Connell, 2002; O’Connell et al., 2001) and the guanosine triphosphatase activating protein (GAP) CYK-4 (Jenkins et al., 2006) are all required for embryogenesis. However, SPE-11 is the only known C. elegans pro-

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