Anucleate Caenorhabditis elegans sperm can crawl, fertilize oocytes and direct anterior-posterior polarization of the 1-cell embryo

Although they are both products of meiosis, sperm and oocytes are highly differentiated cells with extremely different attributes. Oocytes are large, sedentary, and nutrient-rich whereas mature haploid sperm are small, motile, and streamlined for motility and fertilization. These differences between the gametes are reflected in the nature of their respective meiotic divisions. Oocyte meiosis yields only a single, large gamete since chromosomes corresponding to three of the four meiotic products are discarded in small karyoblasts, otherwise known as polar bodies (Fig. 1A). In striking contrast, sperm meiosis results in the production of four functional gametes, each of which is at least four times smaller than the original spermatocyte. This down-sizing of the male gametes is further exacerbated by the jettisoning of excess cytoplasm and non-essential organelles into a residual body during the second meiotic division (Fig. 1B).

During fertilization, these two distinctive gametes fuse for the dual purposes of restoring the somatic chromosome number and initiating the development of a new individual (Fig. 1). However, before the genetic contributions of the two gametes can be combined, and the one-cell embryo can function as a pluripotent zygote, several preparatory events must occur. In the nematode, Caenorhabditis elegans, these preparatory events include resumption and completion of oocyte meiotic divisions, secretion of an eggshell, post-meiotic restructuring of the ultra-condensed sperm chromatin, and formation of the oocyte and sperm pronuclei (Fig. 1A). These early events are followed by a round of DNA synthesis and the subsequent migration and joining of the pronuclei (Edgar and McGhee, 1988; Albertson, 1984). Actual mixing of the two genomes first occurs only during mitotic metaphase.

The sheer abundance of genes in C. elegans which can mutate to yield a maternal effect lethal phenotype reveals the large number of maternal products which direct either the process of oocyte differentiation or the subsequent early development of the embryo (for reviews see Kemphues, 1988; Bowerman, 1998). In contrast, only a single paternal effect lethal gene in C. elegans has been well characterized to date (Hill et al., 1989; Browning and Strome, 1996), despite the fact that C. elegans sperm is known to play several essential roles during early development. For instance, fertilization by sperm is essential for both the completion of oocyte meiosis and eggshell secretion. Later, the sperm not only contributes a haploid genome to the developing embryo, but it also provides the embryo with an initial centriole-containing centrosome which subsequently duplicates, expands and eventually forms the first mitotic spindle (Albertson, 1984). More recent work has shown that a C. elegans sperm component, most likely the sperm pronucleus or an associated component, both directs post-meiotic cytoplasmic rearrangements within the 1-cell embryo and specifies the anterior-posterior (A-P) embryonic axis (Goldstein and Hird, 1996).

Although clearly important, the subcellular and molecular details of the sperm’s contributions to these cellular processes remain largely unknown. In this paper, we therefore address the basic but important question of whether the presence of either spermatid DNA or a sperm pronucleus is essential for spermiogenesis, egg activation, or specification of the anterior-posterior embryonic axis in the nematode C. elegans. In this context we consider sperm DNA not only as a possible template for mRNA production but also as a physical entity which is capable of serving as a positional cue for the generation of cellular asymmetries. Nuclear-associated microtubule complexes have been reported to perform polarizing functions in specifying the dorsal-ventral axis in Drosophila oocytes (Roth et al, 1999), facilitating polarized secretion within T killer cells (Sedwick et al., 1999), and regulating pseudopodial activity in fibroblasts (Bershadsky and Vasiliev, 1993). However there are also counterbalancing examples of crawling cells which can retain their polarity in the absence of a nucleus (Malwista and Chevance, 1982). In this paper we present our analysis of both sperm morphogenesis in the absence of sperm DNA and early embryonic development in the absence of a sperm pronucleus. We demonstrate that anucleate sperm are able to carry out several normal sperm functions including spermiogenesis, fertilization, egg activation, and A-P axis specification.

Nematodes were cultured using standard culture techniques (Brenner, 1974). All mutant strains were derivatives of the wild-type strain C. elegans var. Bristol. The following strains were used: N2 (wild type), him-5 (e1490) V (Hodgkin et al., 1979), emb-27 (g48ts) II (Cassada et al., 1981), emb-30 (g53ts) III (Cassada et al., 1981), and fem-1 (hc17ts) IV (Nelson et al., 1978).

Spermatids were isolated from the gonads of celibate males and subsequently activated in vitro as previously described (Ward et al., 1983). For spermatid activation studies, spermatids from celibate males were dissected in a drop of sperm medium between two lines of vacuum grease and covered by a coverslip. Medium containing activator (200 μg/ml Pronase E) was then added to one side of the slide and wicked through to replace the original medium which lacked activator.

To score the percentage of anucleate spermatids, male gonads were dissected in sperm medium containing 100 μg/ml lipid soluble, DNA binding Hoechst dye 33342 (Sigma). To prevent cellular damage, the cells were first analyzed using Nomarski/DIC optics before illuminating the specimens with UV epifluoresence to visualize the Hoechst dye.

Paternal effect embryos were created by crossing feminized fem-1 hermaphrodites to him-5 (e1490ts) males containing either the emb-27 (g48ts) or emb-30 (g53ts) mutation. The him-5 (high incidence of males) genetic background ensured a plentiful supply of males for both genetic crosses and analysis of spermiogenesis, and the presence of him-5 mutations did not alter either the emb-27 or emb-30 sperm phenotypes. To produce fem-1 ‘females’ for these crosses, fem-1 (hc17ts) animals were up-shifted from the permissive temperature of 16°C to the restrictive temperature of 25°C as L1 larvae. emb-27 and emb-30 males were up-shifted as L2 rather than L1 larvae, which increased mating success without decreasing the percentage of anucleate sperm. Because emb-27 (g48ts) males produce a higher percentage of anucleate sperm, the bulk of our paternal effect embryos experiments employed emb-27 crosses. In each case, these results were subsequently confirmed in emb-30 crosses.

A field of young embryos from mated fem-1 hermaphrodites were isolated as follows. To enable visualization of multiple young embryos within a single microscope field, three mated Fem hermaphrodites were picked into 3 μl of embryo buffer (Boyd et al., 1996) which had been deposited on a microscope slide to the right of a pre-cut agar pad. The mated Fem hermaphrodites were cut to extrude the gonadal arms and uterus, and the isolated gonad and embryos within were then transferred to the agar pad using a drawn out capillary pipette and mounted for viewing with Nomarski/DIC optics as described by Sulston et al. (1983). Depending on the experiment, 1-cell embryos, dissected spermathecas, or fields of sperm were either photographed or video-taped using a CCD videocamera and recorded on a time-lapse recorder.

For the analysis of early embryos, a 27.5 gauge needle was used to dissect hermaphrodites in buffer (Boyd et al, 1996) directly on polylysine-subbed slides. Samples were prepared for P-granule immunofluorescence according to Strome and Wood (1982) with minor modifications. The embryos were freeze-cracked and then fixed in −20°C methanol for 15 minutes followed by 5 minutes in −20°C acetone. The slides were air-dried and then blocked in phosphate-buffered saline (PBS) containing 0.5% BSA and 0.1% Tween 20. The slides were incubated at 4°C overnight in anti-P-granule antibody (OIC1D4), washed, and subsequently incubated at 4°C for 6 hours in secondary antibody. After washing and DAPI staining, the slides were mounted using Anti-Fade (Molecular Probes). Immunostaining for PIE-1 was similar to that of P-granules but employed an ethanol rehydration series instead of an air-drying step, and the samples were incubated with secondary antibody for 2 hours at room temperature. Samples were prepared for PAR-3 immunofluorescence according to the method of Etemad-Moghadam et al. (1995) with minor modifications. Freeze-cracked embryos were fixed in −20°C methanol for 15 minutes and washed sequentially in PBT (PBS + 0.1% Tween 20) and PBS. Samples were incubated in primary and secondary antibodies for 1 hour each at 37°C. For tubulin immunostaining (protocol modified from Albertson, 1984), freeze-cracked embryos were fixed in −20°C methanol for 1 hour and blocked for 1 hour at room temperature. The slides were then incubated with a 1:100 dilution of FITC direct-labeled anti-alpha tubulin (DM1A, Sigma) for 2 hours at room temperature. DAPI was added during the last 15 minutes. The slides were washed once in PBS and mounted with Anti-Fade.

To analyze the distribution of SPE-11 within spermatids, 5-10 males were dissected in buffer on a subbed slide and covered with a coverslip. Gentle pressure was used to flatten the sperm into a monolayer, and the coverslip was then removed after freeze-cracking the slide in liquid nitrogen. The cells were fixed for 10 minutes in −20°C methanol followed by 10 minutes in −20°C acetone. At this point, the samples were further processed as previously described (Browning and Strome, 1996).

In order to test whether the sperm nucleus is critical for either the final stages of sperm development or the initial stages of embryonic development, we needed a reliable source of anucleate sperm. In theory, such sperm could be generated through either physical enucleation or laser ablation, but these methods are impractical given the small size of C. elegans sperm and the likelihood that such operations would simultaneously destroy the closely associated sperm centrosome. In addition, the necessary in vitro fertilization methods have yet to be developed for C. elegans. However, our analysis of two chromosome segregation mutants, emb-27 (g48ts) and emb-30 (g53ts), revealed that these temperature-sensitive mutants might provide a source of anucleate spermatids. Although these two mutants were originally isolated as maternal effect lethal mutants (Cassada et al., 1981; Denich et al., 1984), studies by both Fiebach (1989) and ourselves have suggested that the emb-27 and emb-30 gene products are also required paternally. While investigating the primary mutant defect during meiotic chromosome segregation, we found that at the non-permissive temperature of 25°C, emb-27 males produce 97% anucleate spermatids (n=119 spermatids), and emb-30 males produce 81% (n=134) anucleate spermatids. Anucleate spermatids were scored using the DNA dye Hoechst 33342, which is sufficiently sensitive to detect the presence of a single chromosome fragment within spermatids (Fig. 2A-D). The highly condensed chromatin masses are also easily detectable under Nomarski optics.

In C. elegans, spermatocyte meiosis results in the formation of four spherical and sessile spermatids (Fig. 1B). Centered within each of these cells is a highly condensed chromatin mass which is surrounded by an outer shell of electron dense perinuclear material. This perinuclear material is known to include both a single pair of centrioles and RNA (Ward et al., 1981) as well as the spe-11 protein (Browning and Strome, 1996). Technically this combined chromatin mass and associated perinuclear material is not a nucleus because it lacks a nuclear envelope (Ward et al., 1981). However for the sake of simplicity, we will refer to sperm which lack this chromatin mass as ‘anucleate’ sperm. For these sperm to function as motile gametes, they must first undergo a process of cellular morphogenesis (spermiogenesis) which transforms them from sessile, apolar spermatids into motile, bipolar spermatozoa. Nematode spermatozoa are unusual in that they lack flagella and instead crawl via a pseupodial projection (Roberts and Stewart, 1995).

To test whether the sperm chromatin mass is required either as a template for transcription or as a cue for polarization during this cell morphogenetic process, we attempted to activate the anucleate spermatids using previously described methods for in vitro sperm activation (Ward et al., 1983). When celibate mutant males were dissected in activating medium 79±5% of emb-27 (g48ts) and 81±5% of wild-type spermatids activated to form morphologically normal, polarized, crawling spermatozoa (Fig. 2E-G). Comparable results were obtained for emb-30 (g53ts). The activation process itself seems to be normal, taking 5-10 minutes as previously reported for wild-type sperm (Ward et al., 1983). These results dramatically confirm the widely accepted but previously unproved premise that the morphogenetic process of C. elegans spermiogenesis can occur in the complete absence of new transcription. In addition, these results demonstrate that the physical presence of the sperm chromatin mass is required neither to cue nor to stabilize the polarization of C. elegans spermatozoa.

In order to function, C. elegans spermatozoa must crawl directionally from the hermaphrodite vulva (the site of insemination) to the spermatheca (the site of both fertilization and sperm storage). To test whether anucleate sperm can activate in vivo and migrate to the hermaphrodite’s spermatheca, emb-27 and emb-30 mutant males were mated with feminized fem-1 ‘females’ which lack sperm of their own. Mated fem-1 animals were examined for the presence and location of the male sperm. Abundant, crawling, anucleate spermatozoa were detected within the spermathecas of mated fem-1 ‘females’ using Nomarski/DIC optics (Fig. 2H, complementary data for emb-30 not shown). The anucleate spermatozoa were able to both migrate to the spermatheca and also stay there. Space limitations within the female (or hermaphrodite) somatic gonad are such that without persistent ‘homing’ behavior by individual spermatozoon towards the spermatheca, passing oocytes and embryos will quickly and forcibly squeeze any defective spermatozoa out of the spermatheca, into the uterus and out through the vulva (Argon and Ward, 1980). Thus the stable presence of these anucleate sperm within the spermatheca indicates that the sperm nucleus is also unnecessary for this persistent ‘homing’ behavior.

Although the unfertilized eggs of many organisms can be artificially activated by electrical stimulation or changes in calcium levels (Ozil, 1990), egg activation in C. elegans appears to require fertilization by sperm. In wild-type C. elegans hermaphrodites, maturing oocytes within the proximal gonadal arm pause in diakinesis of meiotic prophase I until they are positioned immediately proximal to the spermatheca (McCarter et al., 1999). As the oocytes are ovulated, they re-enter the cell cycle with one of two possible fates. Those that are subsequently fertilized within the spermatheca are stimulated to adopt an oblong cell shape, secrete an eggshell, and complete both meiotic divisions. In contrast, those that remain unfertilized neither secrete an eggshell nor complete meiosis, but, instead, undergo multiple rounds of non-productive, endomitotic DNA synthesis in the absence of cytokinesis (Fig. 1, also see Ward and Carrel, 1979). Thus, although no methods currently exist to extensively test artificial activation methods in these singly and internally ovulated C. elegans oocytes, fertilization by sperm may be essential for their choice of meiotic completion and eggshell formation rather than endomitosis.

Evidence that a sperm-contributed factor is essential for normal egg activation stems from earlier analysis of the paternal effect mutant, spe-11. In these studies, wild-type oocytes fertilized by spe-11 sperm produced weak eggshells and exhibited defects in oocyte meiotic divisions (Hill et al., 1989). Because SPE-11 is normally localized to the perinuclear material which surrounds the sperm chromatin mass (Browning and Strome, 1996), we wondered whether SPE-11, in the absence of sperm chromatin, could still segregate to spermatids during spermatocyte meiosis and later function in egg activation. To test for the presence of SPE-11 in anucleate spermatids and/or to discover its localization pattern within anucleate spermatids, isolated gonads from wild-type and restrictively grown emb-27 males were immunostained using anti-SPE-11 antibody. During meiotic cell divisions of both wild-type and emb-27 males, SPE-11 segregated to the spermatids. However, while SPE-11 in wild-type spermatids was localized specifically to the perinuclear material (Fig. 3A,B), SPE-11 in anucleate emb-27 spermatids was distributed throughout the cytoplasm in a speckled, granular pattern (Fig. 3C,D). In the rare emb-27 sperm that do contain chromatin, SPE-11 surrounds the chromatin mass, which indicates that SPE-11’s affinity for sperm chromatin (or the perinuclear material) is unaltered in emb-27 mutants.

To subsequently test whether anucleate sperm with abnormally localized SPE-11 can fertilize and activate oocytes, mutant emb-27 males were crossed with fem-1 ‘females’. The anucleate spermatozoa proved not only to fertilize oocytes but also to fully activate them. One hundred percent (52/52) of embryos derived from these crosses formed a normal eggshell as demonstrated by their full osmotic resistance to de-ionized water. Furthermore, DAPI analysis of such embryos showed that, of the 1-cell embryos that were scored early enough to ensure that they lack a sperm pronucleus, 60/60 underwent normal oocyte meiotic divisions and produced two meiotic polar bodies. As in control embryos, the polar bodies were positioned with one polar body located just under the outer eggshell and the other under the inner chitin-containing membrane (for a description of nematode eggshell structure, see Wharton, 1983). Similar results were obtained in crosses between fem-1 ‘females’ and emb-30 males.

Following the completion of oocyte meiosis, the oocyte pronucleus forms, undergoes a single round of DNA synthesis, and then migrates posteriorly to meet the sperm pronucleus (Fig. 1). In a process called centration, the joined but unfused pronuclei then migrate back to the center of the embryo prior to nuclear envelope breakdown and the onset of mitotic metaphase. This migration of the oocyte pronucleus has been shown to be mediated, in part, by the sperm centrosomes which arise from the duplicated and activated sperm-contributed centriolar pair (Strome and Wood, 1983; Albertson, 1984). However, because the mutant centrosomes were not embedded within a normal chromatin-perinuclear complex during the spermatid/spermatozoon phase of their existence, it was possible that their subsequent functioning might be adversely affected; a change which, in turn, could impact on sperm aster formation and consequently the path and timing of oocyte pronuclear migration.

To analyze the position and timing of pronuclear migration in oocytes fertilized by anucleate sperm, the dynamics of these movements were captured both on time-lapse videos and in photographic time series (Fig. 4). In the absence of a sperm pronucleus, the oocyte pronucleus in fem-1 × emb-27 embryos behaved the same as in wild-type controls: the oocyte pronucleus migrated slowly toward the embryonic center in an initial slow phase, then moved rapidly to its posterior-most migration point (Fig. 4A,B,G,H), before moving back to the embryonic center (Fig. 4C,I) and entering mitotic metaphase (Fig. 4D,J). In addition, the position and timing of events in the ‘healthiest’ 40% of the video-taped mutant embyros (13/33) were indistinguishable from those in wild-type controls (Fig. 4; Table 1). Healthy embryos were defined as those that subsequently underwent an apparently normal first asymmetric cell division. Note, however, that early pronuclear migration events occurred normally in a full 76% of the videotaped embryos. The other 12/33 (36%) embryos were considered ‘less healthy’ only because they developed cytokinesis defects during the last part of the first mitotic division (see below).

Although many organisms have one or more of their embryonic axes predetermined during oogenesis, C. elegans oocytes, like those of ctenophores and mammals, lack a pre-determined axis (for review, see Wall, 1990). The A-P axis is the first to be established; the redistribution of A-P markers occurs during a period of cytoplasmic streaming which follows the completion of oocyte meiosis (Hird and White, 1993; Hird et al., 1996). In wild-type 1-cell embryos, the oocyte and sperm pronuclei invariably mark the respective anterior and posterior ends of the embryo. However, by using experimental conditions to alter the site of sperm entry, Goldstein and Hird (1996) convincingly established that the embryonic end closer to the point of sperm entry always becomes the embryonic posterior. In addition, they showed that the ‘fountainhead’ pattern of cytoplasmic streaming (internal streaming towards the posterior and simultaneous cortical streaming back towards the anterior) is actually directed towards the sperm pronucleus or an associated component. These important results raised the possibility that the sperm pronucleus itself might be essential for both directed cytoplasmic streaming and the establishment of A-P polarity.

In wild-type embryos, cytoplasmic streaming initially begins as the pronuclei first become visible (Goldstein and Hird, 1996), but it is soon accompanied by actin-based waves of cortical contractions (Fig. 5A) which culminate in the formation of a deep central pseudocleavage (PC) furrow during the initial stages of the oocyte pronucleus’ posterior migration (Fig. 4A; Nigon et al., 1960). Shortly after the oocyte pronucleus migrates into the posterior half of the embryo, the pseudocleavage furrow and all other cortical contractions regress. The stereotypic, cortical contractions provide a useful, photographable marker of cytoplasmic streaming since the contractions and streaming processes are temporally linked. On the other hand, the biological significance of these cortical contractions is uncertain since embryos from nop-1 mothers undergo cytoplasmic streaming and are perfectly viable even though they fail to form pseudocleavage furrows (Rose et al., 1995). Thus while the cortical actin network itself is required for both streaming and A-P polarization (Hill and Strome, 1990; Hird and White, 1993), the large scale actin-based cortical contractions are dispensable (Rose et al., 1995).

To formally test whether the sperm pronucleus is required for either cytoplasmic streaming or the segregation of A-P polarity markers, we examined these events in oocytes that had been fertilized by anucleate sperm. Video analysis of such embryos fathered by either emb-27 or emb-30 males revealed that cytoplasmic streaming, cortical contractions, and the formation of a pseudocleavage furrow all occur normally in the absence of a sperm pronucleus (Fig. 5). We then examined the localization patterns of three distinct markers of A-P polarity using immunofluorescence. Note that because we could not use the sperm pronucleus as a marker for the posterior end, we instead used the position of the meiotic polar bodies as a marker for the anterior end. Antibodies against germline-specific RNA-protein complexes known as P-granules revealed a localization pattern similar to that in wild-type embryos; P-granules were uniformly distributed throughout the cytoplasm of meiotic 1-cell stage embryos fathered by emb-27 males (Fig. 6A,B) but segregated to the posterior during cytoplasmic streaming and remained there for the remainder of the first cell cycle (Fig. 6C,D). As in wild-type embryos, P-granules continue to segregate to the posteriorly positioned germline progenitor cells in subsequent cell divisions. Terminal stage multi-cellular embryos from fem-1 × emb-27 crosses contain P-granules in 1-3 posterior blastomeres (Fig. 6E,F; Table 2), but fail to gastrulate and eventually arrest development with 200±50 cells. In wild-type embryos, P-granules segregate to the single P4 germline progenitor cell which then migrates internally during gastrulation and subsequently divides one more time during embryogenesis to form the two germline progenitor cells, Z2 and Z3. Similar results were obtained in emb-30 crosses (data not shown). In all paternal effect experiments, only embryos that completely lacked sperm DNA were used in our analysis.

The transcriptional silencer PIE-1 was used as a second marker of A-P polarity. In wild-type embryos, PIE-1 is initially distributed uniformly throughout the cytoplasm but segregates to the embryonic posterior by the time of pronuclear meeting (Tenenhaus et al., 1998). Like P-granules, PIE-1 continues to segregate to germ line progenitor cells during subsequent cell divisions, however PIE-1 serves as a unique A-P marker since it is not strictly a component of the large P-granule complex (Tenenhaus et al., 1998). In embryos fertilized by anucleate emb-27 sperm, PIE-1 segregation patterns were essentially normal: PIE-1 was initially distributed throughout the cytoplasm during the completion of oocyte meiosis (Fig. 6G), it then segregated to the embryonic posterior as the oocyte pronucleus migrated to the posterior end (Fig. 6H), and it continued to segregate to posterior blastomeres in subsequent cell divisions (Fig. 6I). Consistent with PIE-1 localization patterns in wild-type embryos (Tenenhaus et al., 1998), pie-1 protein levels dropped precipitously in older embryos (data not shown).

As a counterbalancing marker of anterior polarity, we examined the distribution of PAR-3, a maternally contributed protein which is believed to function as part of the polarization machinery per se (Kemphues et al., 1988, Cheng et al., 1995; Etemad-Moghadam et al, 1995; Kemphues and Strome, 1997). In embryos fertilized by anucleate emb-27 sperm, PAR-3 exhibited a seemingly wild-type localization pattern. PAR-3 was uniformly distributed in meiotic 1-cell embryos (Fig. 6J,KI) but became localized to the anterior cortex following the completion of oocyte meiosis (Fig. 6L-O).

Since early embryonic development in C. elegans is characterized by a series of asymmetric cell divisions, successful polarization of the zygote can also be assessed by examining the timing and cleavage patterns of the first two cell divisions. In wild-type embryos, the initially symmetric first mitotic spindle shifts posteriorly resulting in the formation of a larger anterior and smaller posterior blastomere (Fig. 4D; Albertson, 1984). Video-tape analysis of live embryos under Nomarski/DIC optics revealed a similar posterior shift of the mitotic spindle in 88% (15/17) of the embryos fertilized by anucleate emb-27 sperm (also Fig. 4J). Although eight of these fifteen embryos exhibited subsequent cleavage defects (see below), seven others divided normally, forming a large anterior and smaller posterior blastomere at the 2-cell stage (Fig. 4E, K). Normal asymmetric first cell divisions were also observed in some embryos fertilized by anucleate emb-30 sperm (data not shown). Consistent with these observations, tubulin/DAPI immunostaining of similar 2-cell embryos reveals that, as in wild-type embryos, the two blastomeres differ from each other in both their cell cycle timing and the orientation of their mitotic spindles during the next cell division (Fig. 7, equivalent emb-30 data not shown).

In C. elegans, the oocyte meiotic spindle lacks centrioles, and thus it is the spermatozoon which provides the embryo with a mitotic centrosome (Albertson, 1984; Albertson and Thomson, 1993). In spermatozoa, the single centrosome lies quiescent within the perinuclear material (Ward et al., 1981). Even after fertilization, the sperm centrosome remains quiescent while oocyte meiosis is completed on barrel-shaped, acentriolar meiotic spindles (see Albertson and Thomson, 1993 for analysis of oocyte meiotic spindles). Based on cell cycle studies in Xenopus embryos (Lacey et al., 1999), the single C. elegans sperm centriole pair probably duplicates during the post-meiotic cell cycle transition into S-phase, just after the formation of the pronuclei. Using DAPI/tubulin immunostaining as an assay for microtubule nucleation, we could only first detect active sperm-contributed centrosomes in 1-cell wild-type embryos during late S-phase at which point the two, closely opposed sperm asters were positioned on the cortical side of the sperm pronucleus (Fig. 8A). Although the centrosomes contributed by emb-27 sperm might be predicted to behave differently due to their lack of contact with sperm chromatin during the spermatid/spermatozoon phase of their existence, we found essentially no differences in the timing of sperm centrosome activation. In both wild-type and mutant embryos, tiny asters were only first seen during late S-phase after the centrosome had already duplicated (Fig. 8A,E,F).

During the early part of transitional prophase, these tiny nucleating sperm asters expand and separate from one another. During this time, the asters normally stay in close contact with the sperm pronucleus as they separate and expand (Fig. 8B); in the absence of a sperm pronucleus, the asters separate and expand but remain closely associated with the cell cortex (Fig. 8G,H). During the pronuclear meeting stage when the asters normally are positioned at the junction of the two pronuclei (Fig. 8C), the asters in these paternal haploid embryos were found on opposing sides of the unpaired oocyte pronucleus (Fig. 8I,J). How these asters move from the cortex and capture the oocyte pronucleus remains unclear since the relative speed of these events prevented us from catching re-localizing sperm asters in immunostained specimens. However both our video (Table 1) and tubulin immunofluorescence data suggest that the mutant sperm asters link up with the lone, posteriorly migrated oocyte pronucleus at the same egg-length position as the wild-type asters join the oocyte pronucleus. Since sperm asters migrating along the cortex have to travel a greater total distance to reach the same egg length position as asters migrating around a sperm pronucleus, it is probably not the physical distance migrated by individual sperm asters but rather the oocyte pronucleus and/or another factor in the embryonic posterior which determines the point of pronuclear capture. Our video analysis showed that the rotation of pronuclear-centrosome complex during centration was also relatively unaffected; rotation occurred normally in 100% of the wild-type and 76% (25/33) of the video-taped embryos scored.

Cleavage defects in a subset of embryos fathered by anucleate emb-27 sperm were of two types. In one class of mutant embryos (12/33), cytoplasmic streaming, pronuclear migration, and segregation of polarity markers occurred normally, however the first mitotic division was abnormal due to the formation of third mitotic aster which appeared either immediately prior to or shortly after the breakdown of the pronuclear envelope (data not shown). A second class of mutant embryos (8/33) exhibited numerous early defects which included little or no nucleation of sperm asters. These embryos either failed to cleave altogether or produced weak, incomplete cleavage furrows. However since the primary focus of this paper is on the developmental events which can proceed normally in the absence of a sperm pronucleus, detailed analysis of the microtubule-related defects in these other two classes of mutant embryos will be reported elsewhere (P. L. S. and D. C. S., unpublished data).

The analysis of both spermiogenesis in the absence of sperm DNA and early embryonic development in the absence of a sperm pronucleus have enabled us to test a number of important hypotheses regarding the role of sperm DNA, the sperm chromatin mass, and the sperm pronucleus in these developmental processes. First of all, although earlier biochemical and ultrastructural studies suggested that spermatid DNA was transcriptionally inactive, our data provides the ultimate proof that transcription during spermiogenesis is not required for the morphological conversion of a spherical sessile spermatid into a bipolar, motile spermatozoon. Furthermore, in documenting that these anucleate spermatozoa can not only crawl but also migrate directionally towards the hermaphrodite spermatheca, we have conclusively demonstrated that functional and stable cellular polarity can be achieved in the absence of positional cues from the sperm’s chromatin mass.

Our finding that oocytes fertilized by anucleate sperm can both complete the oocyte meiotic divisions and form osmotically resistant eggshells dramatically demonstrates that the sperm chromatin mass is not required for either of these processes. In addition, these studies demonstrate that although individual components of the perinuclear material may be required for proper egg activation and early embryonic development, the integrity of the perinuclear material as a solid mass surrounding the sperm chromatin may be unnecessary for either the meiotic segregation or the subsequent function of these components. For instance, in the absence of sperm chromatin, the perinuclear component SPE-11 can still function to promote egg activation despite the fact that its normal intracellular localization pattern within the spermatid has been disrupted. This finding is consistent with earlier work showing that functional SPE-11 could be supplied to the embryo either through the oocyte or sperm (Browning and Strome, 1996). Furthermore the remarkably normal early development of embryos fertilized by anucleate sperm suggests that other perinuclear components, including the quiescent sperm centrosome, may continue to function normally during embryogenesis despite their altered intracellular localization patterns.

This is the first published report to describe the early development of C. elegans embryos that lack a sperm pronucleus; previous attempts to physically create such paternal haploids failed since extrusion of the sperm pronucleus through a small hole in the eggshell invariably resulted in a simultaneous loss of the sperm-contributed centrosome (Schierenberg and Wood, 1985). In contrast, maternal haploids have been created previously in three different ways: (1) embryos from mei-1(lf) mothers frequently lack oocyte chromosomes due to meiotic loss within the abnormally large mutant polar bodies (Mains et al., 1990); (2) ovulation defects in ceh-18 mutants lead to the creation of anucleate yet fertilizable oocyte fragments (Rose et al., 1997), and (3) in physical manipulations of wild-type embryos, the oocyte pronucleus can been successfully extruded through a small hole in the eggshell (Schierenberg and Wood, 1985). In the case of the maternal haploids, the embryos arrested prior to morphogenesis but underwent normal early embryonic cell divisions. The results presented here demonstrate that at least some paternal haploids also undergo reasonably normal early embryonic development, and thus we can conclude that early embryonic cell divisions in C. elegans embryos are only mildly affected by embryonic haploidy regardless of the origin of the single remaining pronucleus.

This study also has important implications concerning the cellular mechanisms of C. elegans axis specification. In many organisms, the position of sperm entry is critical for the specification of at least one embryonic axis. For instance, the sperm entry point plays an essential role in the determination of the dorsal-ventral axis in both amphibians (Ancel and Vintemberger, 1948; Nieuwkoop, 1977) and spiralian embryos (Morgan and Tyler, 1930; Luetjens and Dorresteijn, 1998). In C. elegans and other closely related nematodes, the sperm specifies the posterior end of the embryos, although this mechanism of determining A-P polarity is shared neither by all male/female nematode species (Goldstein et al., 1998) nor the numerous nematode species which develop parthenogenically. However for the species in which the sperm does specify an embryonic axis, what is the underlying cellular mechanism? In Xenopus, the sperm asters normally control the 30° rotation of the cell cortex relative to the rest of the egg mass, but since this effect of the sperm can be overridden by gravity, the rotation itself is thought to be the key signaling event (Vincent and Gerhart, 1987). In C. elegans, the development of A-P polarity is apparently linked to a period of cytoplasmic rearrangement which culminates in the partitioning of both P-granules and PIE-1 to the embryonic posterior and the asymmetric distribution of the various par proteins to either the anterior or posterior cortex (Kemphues and Strome, 1997). This cytoplasmic streaming is normally directed towards the sperm pronucleus or an associated component, and unlike the situation in Xenopus, gravity has no effect on axis specification in C. elegans (Goldstein and Hird, 1996). In the present studies, we have ruled out an essential role for the sperm pronucleus in either cytoplasmic streaming or in A-P polarization.

If the sperm pronucleus is not the critical polarizing factor, other possibilities include the sperm centrosome, a localized cortical change induced by sperm entry, or a diffusible cytoplasmic factor. Current evidence that the sperm asters might play such a role is mixed. Microfilaments rather than microtubules have been shown to be essential for A-P polarization (Strome and Wood, 1983; Hill and Strome, 1990), and treatment of embryos with microtubule inhibitors does not prevent the polarization of A-P markers (Strome and Wood, 1983). However, the microtubule inhibitors may not completely depolymerize microtubules, and alterations of the microtubule cytoskeleton can cause cells that normally divide symmetrically to undergo cytoplasmic streaming and divide asymmetrically (Hird and White, 1993). Alternatively, since embryos first initiate cytoplasmic flow at the time of pronuclear formation (Goldstein and Hird, 1996) and before any significant sperm aster expansion, perhaps it is the sperm centrioles and/or associated factors rather than fully formed microtubule asters which either trigger or cue cortical flow at the sperm entry point. If correct, this model predicts that embryos that lack a sperm-contributed centrosome will be both unable to cleave (Schierenberg and Wood, 1985) and unable to establish an A-P axis. Providing definitive evidence to support a polarization role for either the sperm centrioles or the sperm asters will require, in part, the identification and analysis of paternal effect mutants which either lack sperm centrioles or are unable to support centriole function. Investigating this role in C. elegans sperm should be easier than in most organisms, since the sperm centrosome is not required for the sperm’s unique non-actin, non-tubulin based cell motility (Roberts and Stewart, 1995). Recent studies in our own lab suggest that a subset of our chromosome segregation mutants may, in fact, have defects in the segregation of additional sperm components. Thus these mutants should provide a useful tool for testing the requirement of either the sperm centrioles or other critical sperm-contributed factors in the specification of the A-P axis (P. L. S. and D. C. S., unpublished data).

We would like to dedicate this paper to the memory of Jay Weems, a graduate student who was fascinated by the biology of sperm-oocyte interactions and who enthusiastically encouraged both of us during the early stages of this work. We also thank S. Strome, K. Kemphues, and G. Seydoux for antibodies, and A. Golden, L. Wille, and G. Holt for reading versions of the manuscript. In addition, we thank R. Cassada for stimulating discussions and encouragement during early stages of this work. The work was supported by grants from NSF (IBN 92653092) and the Jeffress Memorial Trust to D. C. S., as well as Sigma Xi and Houston Livestock and Rodeo awards to P. L. S. Some of the strains used in these studies were obtained from the Caenorhabditis Genetics Center, which is supported by the NIH National Center for Research Resources (NCRR).