355
Development 127, 355-366 (2000)
Printed in Great Britain © The Company of Biologists Limited 2000
DEV3070
Anucleate Caenorhabditis elegans sperm can crawl, fertilize oocytes and
direct anterior-posterior polarization of the 1-cell embryo
Penny L. Sadler1,2 and Diane C. Shakes1
1Department
2Department
of Biology, College of William and Mary, Williamsburg, VA 23187, USA
of Biology, University of Houston, Houston, Texas, USA
*Author for correspondence (e-mail: [email protected])
Accepted 1 November 1999
SUMMARY
It has long been appreciated that spermiogenesis, the
cellular transformation of sessile spermatids into motile
spermatozoa, occurs in the absence of new DNA
transcription. However, few studies have addressed
whether the physical presence of a sperm nucleus is
required either during spermiogenesis or for subsequent
sperm functions during egg activation and early zygotic
development. To determine the role of the sperm nucleus
in these processes, we analyzed two C. elegans mutants
whose spermatids lack DNA. Here we show that these
anucleate sperm not only differentiate into mature
functional spermatozoa, but they also crawl toward and
fertilize oocytes. Furthermore, we show that these
anucleate sperm induce both normal egg activation and
anterior-posterior polarity in the 1-cell C. elegans embryo.
The latter finding demonstrates for the first time that
although the anterior-posterior embryonic axis in C.
elegans is specified by sperm, the sperm pronucleus itself is
not required. Also unaffected is the completion of oocyte
meiosis, formation of an impermeable eggshell, migration
of the oocyte pronucleus, and the separation and expansion
of the sperm-contributed centrosomes. Our investigation of
these mutants confirms that, in C. elegans, neither the
sperm chromatin mass nor a sperm pronucleus is required
for spermiogenesis, proper egg activation, or the induction
of anterior-posterior polarity.
INTRODUCTION
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
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
Key words: Spermiogenesis, Fertilization, Egg activation,
Caenorhabditis elegans, Sperm pronucleus, Axis specification,
Paternal effect, emb-27, emb-30, Centrosome
356
P. L. Sadler and D. C. Shakes
Endomitosis in
absence of fertilization
Oocyte Maturation
Oocyte
Budding
Meiotic One Cell Stage
NEBD
Oocyte
Growth
Oocyte
Metaphase I
Spindle
Ovulation
Oocyte
Metaphase II
Spindle
NERF
Diakinesis
of Prophase I
Fertilization
Polar Bodies
Sperm
Sperm DNA
Completion
Meiosis I
Mitotic One Cell Stage
PN
Migration
PN
Meeting
PN Rotation
A
Meiosis II
Chromosome
Condensation
P
Transitional Prophase
NEBD
Posterior Shift
Bipolar Spindle
Chromosome
Separation
A
Metaphase
Anaphase
Sperm PN &
Centriole Pair
S Phase
Interphase
Sperm Aster
Expansion
PN Centration
Chromosome
Congression &
Alignment
Oocyte PN
Centriole
Duplication
& Separation
Two Cell Stage
Cleavage Furrow
Ingression
NERF
AB
P1
Telophase/Cytokinesis
Spermatogenesis
Fig. 1. Spermatogenesis and stages
in the development of 1-cell
embryos. (A) Development of 1Residual
Completion
Body (1or 2)
cell embryos. Immediately prior to
Meiosis I
ovulation, mature C. elegans
oocytes leave their extend
Spermiogenesis diakinetic pause and undergo
or
nuclear envelope breakdown
Sperm Activation (NEBD). Ovulated but unfertilized
o
1
oocytes undergo endomitotic
Spermatocyte
rounds of DNA synthesis; whereas
fertilized and activated oocytes
Budding
Spermatids
o
2
Crawling
Spermatids
secrete an eggshell and complete
(1N)
Spermatocytes
Spermatozoon
two rounds of meiotic divisions. At
each division, one chromosome set
is discarded within a small karyoblast known as a polar body. Remodeling of the condensed sperm and oocyte chromosomes occurs only after
the completion of oocyte meiosis as the chromatin decondenses and nuclear envelopes reform (NERF) around each chromosome set.
Pronuclear (PN) formation marks the post-meiotic entry into interphase when both pronuclei undergo a round of DNA synthesis, and the sperm
centriolar pair duplicates. Completion of S-phase and entry into prophase is marked by condensation of the chromosomes and enlargement of
the pronuclei. An unusual, extended transitional prophase follows which encompasses the events of PN migration, PN meeting, and the
subsequent rotation/centration of the PN/centrosome complex. Mitosis begins as the pronuclear envelopes breaks down (NEBD) and a bipolar
mitotic spindle forms. Later, as the first cell division nears completion, nuclear envelopes reform (NERF) around each set of chromosomes, and
the embryo divides asymmetrically to form a large, anterior AB cell and a smaller, posterior P1 cells. (B) Mature primary spermatocytes
separate from the syncytial germline prior to diakinesis of meiosis I. Meiosis I ends with a complete or incomplete actin-myosin based cell
division to form two secondary spermatocytes (Ward et al., 1981). The second meiotic division involves a highly asymmetric, cytochalasininsensitive cell division, called spermatid budding, during which an essential subset of organelles and proteins are selectively partitioned to the
budding spermatids (Ward et al., 1981). During the final stage of spermatogenesis, sessile spermatids are transformed into motile, bipolar
spermatozoa.
Meiosis II
X
B
Anucleate C. elegans sperm
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 anteriorposterior 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.
MATERIALS AND METHODS
Nematode strains and culture methods
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).
Spermatid analysis
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.
Genetic crosses and analysis of embryos
Paternal effect embryos were created by crossing feminized fem-1
hermaphrodites to him-5 (e1490ts) males containing either the emb27 (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
357
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.
Immunofluorescence
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 phosphatebuffered 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 AntiFade.
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).
RESULTS
Chromosome segregation mutants provide a useful
source of anucleate spermatids
In order to test whether the sperm nucleus is critical for either
358
P. L. Sadler and D. C. Shakes
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.
Anucleate spermatids undergo cellular
morphogenesis
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
Fig. 2. Cellular morphogenesis (spermiogenesis) and directed motility of anucleate sperm.
(A) Field of wild-type (him-5) spermatids under Nomarski optics. Under Nomarski optics,
the highly condensed chromatin masses appear as centrally located, raised masses.
(B) Same field of cells, viewed with Hoechst to show DNA. (C,D) Equivalent fields of
emb-30 (g53ts) spermatids show that most are anucleate. Rare spermatids containing
DNA are indicated with a <. The elongated appearance of some chromatin masses is due
to movement of some cells during the extended Hoechst photographic exposure. Under
these Hoechst staining conditions, the presence of a single chromosome within the mutant
spermatids can be easily detected. (E) Nomarski image of emb-27 (g48ts) spermatids
isolated from celibate emb-27 males reveals that most are anucleate. Small arrows indicate
rare cells that have DNA; large arrow points to an anucleate cell. (F) Eight minutes after
exposure to activation medium, most of the sperm have undergone cellular morphogenesis
and formed active pseudopods. (G) Same field of cells, stained with Hoechst to show
DNA. Most cells lack DNA, although a few have fragmented DNA (large arrow) or
contorted nuclei. (H) Dissected spermatheca from a fem-1 ‘female’ that had been mated to
emb-27 (g48ts) males. Motile, anucleate spermatozoa can be seen in between the surface
of the oocyte and the remnants of the spermathecal wall. Scale bar in G indicates 10 µm
for E-G; scale bar in H indicates 10 µm for A-D and H.
Anucleate C. elegans sperm
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.
Anucleate sperm can fertilize oocytes and support
the meiotic 1-cell stage
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 reenter 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 nonproductive, 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.
A
B
C
D
359
Fig. 3. SPE-11 immunostaining in wild-type and anucleate
spermatids. Sperm spreads from wild-type (A,B) and emb-27 (C,D)
males, stained with DAPI to visualize the DNA (A,C) or
immunostained with SPE-11 antiserum (B,D). In wild-type
spermatids, SPE-11 surrounds the tightly condensed sperm
chromatin mass in a tight ring (B). Arrowheads show the same cell
stained with DAPI (A) and SPE-11 antibody (B). In anucleate
spermatids, SPE-11 is present in a speckled pattern through the cell
(C,D), see arrow for an example. Note that in the few emb-27
spermatids that have DNA (C, arrowhead), SPE-11 surrounds the
chromatin mass (D, arrowhead).
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.
360
P. L. Sadler and D. C. Shakes
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.
Migration of the oocyte pronucleus does not require
a sperm pronucleus
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).
Establishment of anterior-posterior polarity in the
absence of a sperm pronucleus
Although many organisms have one or more of their embryonic
Fig. 4. Nomarski time series of (A-F) wild-type and (G-L) one of the
more normal emb-27 paternal haploid embryos. (A,G) In
pseudocleavage stage embryos, the oocyte pronucleus is situated in
the middle of the embryos but a posterior-localized sperm pronucleus
(right) can only be seen in the wild-type embryo. The oocyte
pronucleus migrates to a similar posterior position in the presence
(B) or absence (H) of a sperm pronucleus. The pronuclearcentrosome complex then centrates (C,I) and first mitosis (D,J)
ensues. In both wild-type and paternal haploid embryos, the mitotic
spindle is shifted posteriorly (D,J). In 2-cell embryos, the anterior
blastomere is larger than the posterior blastomere (E,K). Wild-type
embryos eventually undergo morphogenesis to form a worm (F)
whereas haploid paternal embryos arrest development as multicellular balls of cells (L). Note that the late stage embryos shown in
(F,L) are not the same embryos as shown in the proceeding time
series. Scale bar for A-E and G-K indicates 12 µm; for F and L,
15 µm.
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
Anucleate C. elegans sperm
Table 1. Position and timing of early events
(A)
Position relative to egg-length
Sperm
Wild type§
Anucleate¶
PC* furrow
PN meeting‡
Centration
55.9±3.6% (n=6)
56.5±6.0% (n=5)
71.7±2.6% (n=6)
74.1±1.7% (n=6)
51.6±2.0% (n=6)
52.6±2.3% (n=6)
(B)
Migration of the oocyte pronucleus (minutes:seconds)
Sperm
Wild type
Anucleate¶
PC to meeting
Meeting to center
0.33±0.10 (n=6)
0.35±0.07 (n=3)
2.19±0.20 (n=6)
2.27±0.43 (n=6)
*PC, pseudocleavage.
‡PN, pronuclear meeting or posterior-most migration point.
§fem-1 × him-5 crosses.
¶fem-1 × emb-27(g48) ; him-5 crosses. Data from sub-set of embryos that
had a normal first asymmetric cell division.
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
Fig. 5. Cortical contractions during cytoplasmic streaming occur
both in the presence (A) and absence (B) of a sperm pronucleus. An
oocyte pronucleus can be seen on the anterior (left) side of both wildtype (A) and emb-27 (B) fathered embryos, but a sperm pronucleus
can be seen near the posterior (right) plasma membrane only in the
wild-type embryo. Scale bar indicates 24 µm.
361
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 germlinespecific RNA-protein complexes known as P-granules revealed
a localization pattern similar to that in wild-type embryos; Pgranules 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
362
P. L. Sadler and D. C. Shakes
Fig. 6. Segregation of
polarity markers in
paternal haploid embryos.
(A,C,E) DAPI-stained
paternal haploid embryos
with corresponding Pgranule immunostaining
(B,D,F). Note the absence
of a sperm pronucleus
(A) and a smaller than
normal, haploid
metaphase plate (C). In a
meiosis II stage embryo
(A), P-granules are
uniformly distributed (B).
By 1st mitosis (C),
P-granules are well
segregated to the
posterior (D). P-granules
continue to segregate to
posterior blastomeres,
and can be seen in a
couple of cells in a late
stage embryo (E,F). PIE1 exhibits a segregation
pattern much like that of
P-granules in equivalently staged embryos:
uniform distribution during meiosis (G),
posterior localization during mitosis (H), and
segregation to a single posterior blastomere in
later stage embryos (I). (J,L,N) DAPI-stained
paternal haploid embryos with corresponding
PAR-3 immunostaining (K,M,O). In meiosis I
stage embryos (J), PAR-3 is difficult to detect
but uniformly distributed (K). During late S
phase (L), PAR-3 is found at the anterior
cortex in a somewhat patchy pattern (M). As
the 1-cell stage progresses, this anterior
localization pattern tightens up (N,O). Scale
bar in A-I indicates 13 µm; in J-O, 15 µm.
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).
Behavior of sperm centrosomes in embryos
fertilized by anucleate sperm
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
Anucleate C. elegans sperm
Table 2. Germline progenitor cells in terminal-stage
embryos
Percentage of P-granule containing cells
Sperm genotype
Wild type
emb-27 (g48)
emb-30 (g53)
0
1
2
>2
n
0
5.5
18.5
0
72
61
100
17
15.5
0
5.5
5
>1000
90
135
363
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 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
DISCUSSION
cortical side of the sperm pronucleus (Fig. 8A). Although the
centrosomes contributed by emb-27 sperm might be predicted
The analysis of both spermiogenesis in the absence of sperm
to behave differently due to their lack of contact with sperm
DNA and early embryonic development in the absence of a
chromatin during the spermatid/spermatozoon phase of their
sperm pronucleus have enabled us to test a number of important
existence, we found essentially no differences in the timing of
hypotheses regarding the role of sperm DNA, the sperm
sperm centrosome activation. In both wild-type and mutant
chromatin mass, and the sperm pronucleus in these
embryos, tiny asters were only first seen during late S-phase
developmental processes. First of all, although earlier
after the centrosome had already duplicated (Fig. 8A,E,F).
biochemical and ultrastructural studies suggested that spermatid
During the early part of transitional prophase, these tiny
DNA was transcriptionally inactive, our data provides the
nucleating sperm asters expand and separate from one another.
ultimate proof that transcription during spermiogenesis is not
During this time, the asters normally stay in close contact with
required for the morphological conversion of a spherical sessile
the sperm pronucleus as they separate and expand (Fig. 8B);
spermatid into a bipolar, motile spermatozoon. Furthermore, in
in the absence of a sperm pronucleus, the asters separate and
documenting that these anucleate spermatozoa can not only
expand but remain closely associated with the cell cortex (Fig.
crawl but also migrate directionally towards the hermaphrodite
8G,H). During the pronuclear meeting stage when the asters
spermatheca, we have conclusively demonstrated that
normally are positioned at the junction of the two pronuclei
functional and stable cellular polarity can be achieved in the
(Fig. 8C), the asters in these paternal haploid embryos were
absence of positional cues from the sperm’s chromatin mass.
found on opposing sides of the unpaired oocyte pronucleus
Our finding that oocytes fertilized by anucleate sperm can
(Fig. 8I,J). How these asters move from the cortex and capture
both complete the oocyte meiotic divisions and form
the oocyte pronucleus remains unclear since the relative
osmotically resistant eggshells dramatically demonstrates that
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 Fig. 7. Cell cycle and spindle orientation differences between the anterior and
posterior blastomere do not require a paternal genome. DAPI-stained wild-type
by anucleate emb-27 sperm were of two types. In (A) and paternal haploid (C) embryos reveal that the anterior AB cell (left) is
one class of mutant embryos (12/33), cytoplasmic further ahead in the cell cycle (anaphase) than the posterior P1 cell (metaphase).
streaming, pronuclear migration, and segregation of Furthermore, due to a rotation of the posterior spindle, tubulin immunostaining of
polarity markers occurred normally, however the these same embryos reveals that the spindles in the anterior and posterior
first mitotic division was abnormal due to the blastomeres lie at right angles to one another in both the wild-type (B) and
formation of third mitotic aster which appeared paternal haploid (D) embryos. Scale bar indicates 20 µm.
364
P. L. Sadler and D. C. Shakes
Fig. 8. Visualization of
centrosome dynamics in wildtype and paternal haploid
embryos which have been
immunostained for tubulin.
(A) In a wild-type embryo just
after the completion of meiosis,
a remnant of the meiotic spindle
can be seen at the bottom left
while the sperm asters are just
beginning to nucleate
microtubules in the posterior
(far right). As the oocyte
pronucleus moves to the
posterior, the sperm asters
separate, expand, and migrate to
the anterior side of the sperm
pronucleus (B). Shortly after
pronuclear meeting and astral
capture, the asters lie in the
junction between the oocyte and sperm pronuclei (C). The pronuclear-centrosome complex must rotate as it centrates such that the mitotic
spindle can set up along the long axis of the embryo (D). (E,G,I) emb-27 paternal embryos immunostained with tubulin antibodies and (F,H,J)
the corresponding DAPI-stained embryos. In S-phase paternal haploid embryos (E,F), a residual meiotic spindle can be seen in the bottom left
while two small sperm asters (posterior cortex at right) are beginning to nucleate asters. During separation, the asters remain close to the cortex
(G,H). The sperm asters later capture the single, un-paired oocyte pronucleus (I,J). Scale bar indicates 20 µm.
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
Anucleate C. elegans sperm
Fig. 9. Schematic summary of events in 1-cell embryos fertilized by
either wild-type (left) or anucleate sperm (right). (A) Post-meiotic,
S-phase embryos during the initiation of cytoplasmic
rearrangements. (B-D) Transitional prophase 1-cell embryos.
(B) Initiation of pronuclear migration is accompanied by polarized
segregation and localization of both PAR-3 and P-granules as well as
by the nucleation, separation and expansion of the duplicated sperm
centrosomes. (C) The oocyte pronucleus reaches its posterior-most
point (with or without a sperm pronucleus present). The oocyte
pronucleus is captured by the sperm asters. (D) The pronuclearcentrosome complex completes rotation and centration along a
predetermined A-P axis. P-granules are shown as green dots, PAR-3
is marked in red, and centrosomes are small black circles.
Microtubules were not drawn in C and D in order to emphasize the
position of the centrosomes relative to the pronuclei.
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
365
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).
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