DEVELOPMENTAL
BIOLOGY
Fertilization
73,304-321
(1979)
and Sperm Competition in the Nematode elegans
Caenorhabditis
SAMUEL WARD AND JOHN S. CARREL Carnegie Maryland
Institution of Washington, Department 21210, and Department of Biological
Received
December
of Embryology, 115 West University Chemistry, Harvard Medical School, 02115
4, 1978; accepted
in revised
form
June
Parkway, Baltimore, Boston, Massachusetts
6, 1979
The process of fertilization by hermaphrodite and male sperm is described. In the hermaphrodite fertilization occurs in the spermatheca by the first sperm to contact the oocyte. Other sperm that contact the oocyte are swept into the uterus but they crawl back into the spermatheca to fertilize subsequent oocytes so that every sperm fertilizes an oocyte. Not every oocyte is fertilized because oocytes are made in excess. Fertilization triggers active movement of oocyte cytoplasmic granules. Sperm penetration is not required for this activation because fertilization-defective mutant sperm trigger activation without penetration. When males copulate with hermaphrodites, their sperm is deposited in the uterus beneath the vulva. These sperm crawl up the uterus to the spermatheca where they displace the hermaphrodite sperm from the spermathecal walls and preferentially fertilize the oocytes. This preferential fertilization appears to be due in part to inhibition of hermaphrodite sperm fertility by the male sperm. The male sperm competition ensures male sperm utilization and thus some outcrossing in a population of predominantly self-fertilizating hermaphrodites. INTRODUCTION
cundity of the hermaphrodite was limited by its production of sperm and that oocytes were produced in excess. Maupas also observed that males were produced in the population at a frequency of about 0.001 but the males he studied were invariably sterile. Maupas’ observations were repeated on a very similar species by Potts (1910), and again on C. elegans isolated near Philadelphia by Honda (1925). Honda was the first worker to obtain males that were fertile. Nigon (1949), in a classic comparative study of nematode cytology and reproduction, established the chromosomal basis of sex determination (hermaphrodites are 5AA,XX; males are 5AA,X) and confirmed the studies of the earlier workers on yet another C. elegans isolate, this one from Bergerac in France. This work was reviewed by Nigon (1965). The work presented here extends these previous studies. By examination of live specimens with light microscopy and fixed
Many specialized gene products must participate in the specific interactions of gametes during fertilization. Some of these gene products will determine the surface topology of the gametes and the specificity of their interaction; others will be required for sperm motility and penetration. In order to identify some of these products and to specify their function, we have begun to isolate and characterize fertilization-defective mutants in the nematode Caenorhabditis elegans (Ward, 1977a; Ward and Miwa, 1978). Before mutants can be studied profitably, it is necessary to understand the normal process of fertilization. A number of studies of C. elegans reproduction have been reported since its isolation some 80 years ago. Maupas (1900) first isolated C. elegans (then called Rhabditis eZegans) in Algeria and described its hermaphroditic mode of reproduction. He discovered that the fe304 0012-1606/79/120304-18$02.00/O Copyright All rights
0 1979 by Academic Press, Inc. of reproduction in any form reserved.
WARD
AND
CARREL
Fertilization
specimens with electron microscopy, we have examined the process of fertilization in more detail than before. We describe the movements of the sperm and the activation of the egg. We also report the phenomenon of male sperm competition, analyze its mechanism, and interpret its significance. These studies provide a detailed background for interpretation of fertilization-defective mutants. MATERIALS
AND
METHODS
Strains and worm culture. Wild-type strain N2 of Caenorhabditis elegans var Bristol was obtained from S. Brenner in 1973. The following mutant strains were E224=dpyemployed: El=dpy-l(e1); ll(e224); E369=unc-51(e369); E51=unc13(e51); E1072=unc-29(e1072); E1035=chel(elO35)fer-l(hc1); HC17=isx-l(hcl7ts); EH66=tra-l(el099)/dpy-18(e364); HCl=ferl(hclts); HC-D4=isx-l(hcl7)dpy-18(e364); E1467=him-l(e1467). E strains are from Brenner’s collection, HC strains from our laboratory. Strains were maintained at 16 or 20°C on NGM plates seeded with E. coli strain OP50 as described by Brenner (1974). Synchronized populations were prepared when needed by collecting worms hatched during a 1-hr interval. Mating experiments. All matings were on 6-cm petri plates of NGM agar seeded at the center with a tiny spot of E. coli 12 hr before mating. Males for mating were picked as juveniles and maintained as virgins for 24 to 48 hr. Ten to 15 males were added with 4 hermaphrodites to each mating plate. For sperm competition experiments hermaphrodites that had laid l-10 zygotes prior to mating were used. After mating for 3-7 hr individual hermaphrodites were transferred to fresh plates at 12to 24-hr intervals and the genotypes of their progeny scored after they had matured. Vital staining. A stock solution of Nile blue A (Allied Chemical) (5% in 95% ethanol) was diluted &fold with M-9 buffer (Brenner, 1974) and 6 to 8 drops were added
and Sperm
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to a 6-cm NGM plate preseeded with E. coli. Males were labeled by placing El467 larvae on a Nile blue A plate. When males had begun to mature they were transferred to a fresh Nile blue A plate for 24-36 hr prior to mating. At the concentration used, the dye does not prevent worm growth or reproduction. Light microscopy. All observations and photographs were made with a Zeiss Universal microscope equipped for Nomarski differential interference contrast observation and epifluorescence. Nile blue A fluorescence was observed with a Zeiss filter combination 486615 which excites with green light and passes red fluorescence for visualization. Nomarski images were recorded on Kodak SO-115 film developed in HCllO dilution D. Fluorescence images were recorded on Kodak Recording Film 2475 developed according to instructions. Identical fields were recorded by interchanging camera backs without altering focus using Planapochromatic lenses to ensure that the focal plane did not change in going from the white light of Nomarski to the red of fluorescence. For observation of live specimens, worms were mounted on slabs of agar under a coverslip as described by Sulston (1976). Uncoordinated worms were usually used, especially the paralyzed E369 because they remain still for time-lapse observation. Photographs were taken with a Zeiss microflash. Detailed studies of fertilization were made from video tapes recorded on a Panasonic NV8030 time-lapse video tape recorder using a Panasonic Nevicon camera. Sperm motility and egg granule activation were recorded at l/18 speed and examined at l/l. Stained preparations were prepared and examined as described previously (Ward and Miwa, 1978). Electron microscopy. Worms were fixed in 1% 0~0~ in 0.08 sodium phosphate (pH 7.4) or 3% glutaraldehyde followed by 0~0~ and prepared for sectioning as described by
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Ward et al. (1975), except that they were cut near the spermatheca in the first fixative to ensure penetration. Sections were examined at 60 or 80 kV with a Zeiss EM10 or JEOL 100s electron microscope. RESULTS
(A) Hermaphrodite
Sperm Development
Light microscopic studies of the development of C. elegans reproductive system were presented by Honda (1925) and again by Nigon (1949, 1965). Hirsh et al. (1976) have described oogenesis with electron microscopy, and Kimble and Hirsh (1979) have described the complete development of the somatic gonad structures. Male sperm development has been described
Oviduct
VOLUME 73,1979
with electron microscopy by Wolf et al. (1978). Hermaphrodite spermatogenesis begins around 24 hr after hatching at 25°C. Primary spermatocytes enter prophase I in the proximal arm of the gonad prior to the gonad’s 180’ turn. By about 35 hr the first spermatocytes have reached diakinesis. The first haploid nuclei become visible at about 39 hr, which corresponds to the time of the last moult. Sperm in the anterior arm of the gonad often mature about 1 hr ahead of those in the posterior arm. Spermiogenesis (the maturation of haploid spermatids) proceeds simultaneously with spermatogenesis from 39 until 48-50 hr. In many adult hermaphrodites fixed and
Spe rmat
heca
Uterus
FIG. I. (a and b) Light micrographs of adult hermaphrodite gonads showing the region of the spermatheca. Orientation is as shown diagrammatically in (c), which shows round spermatids against the oocyte in the oviduct and mature spermatozoa against the wall of the spermatheca. The distal arm of the gonad with oogonia is below in (a) and to the lower left in (b). (a) An older adult with a nearly mature oocyte in the oviduct. x 650. (b) A younger adult showing spermatids in the oviduct and mature spermatozoa in the spermatheca. x 1000. Arrows indicate spermatozoa. Sd = spermatid; 00 = oocyte; Zyg = zygote; SV = spermathecal valve joining the uterus; N = nucleus. Bar = 10 pm.
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Fertilization
stained from 50 to 150 hr after hatching, we observed no new spermatocytes or spermatids, so sperm development occurs only once as the earlier workers showed. Spermatozoa first appear in the ovotestis among the spermatocytes and spermatids. As observed in other nematodes (e.g., Chitwood and Chitwood, 1974, p. 192), the spermatids are round cells that look grainy in the light microscope. As they complete maturation to spermatozoa they become irregular and less grainy (Fig. lb). With the electron microscope it can be seen that this alteration is due to fusion of sperm organ-
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elles called membranous organelles (or special membrane structures) with the plasma membrane and extension of a pseudopod (Fig. 2). This maturation is seen in some males (Wolf et al., 1978) and has been described in several other nematodes (e.g., Foor, 1970; Burghardt and Foor, 1978; McLaren, 1973). Passage of the first mature oocyte pushes sperm from the ovotestis into the spermatheta. Our observations suggest that some sperm crawl in as well. Only mature spermatozoa are found in the spermatheca, and after several oocytes have passed through,
FIG. 2. Electron micrograph of a longitudinal section of the spermatheca corresponding to the view shown in Fig. la. 0~0, fixation. Mature spermatozoa (S) have an electron-opaque nucleus, an electron-dense cytoplasm around the nucleus, and an irregular pseudopod (I?). They are embedded in the wall of the spermatheca. The one spermatid shown in the oviduct in this section has no pseudopod and unfused membranous organelles. In many other sectioned adult hermaphrodites, similar spermatids and no spermatozoa are seen in the oviduct, The nematode’s gut is above the gonad and a zygote in the uterus to the right is badly fixed because of its impermeable eggshell. x 7000.
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only immature spermatids are found in the there, but they do not fertilize. The oocyte ovotestis (now the oviduct) (Fig. 2 and its completes its maturation as its nucleus milegend). It may be that only mature sper- grates from the center of the cell to the end matozoa are carried into the spermatheca distal from the spermatheca (Figs. la and or that spermatids carried into the sper- 3a). This migration is accompanied by spomatheca rapidly complete maturation radic contractions of the oviduct walls and there. contractions of regions of oocyte cytoplasm. The spermatheca is a thick-walled spiral Contractions of the oviduct wall increase tube between the oviduct and the uterus (Figs. 3a and b) and shortly thereafter these (Figs. 1 and 2). It is formed of 24 cells contractions squeeze the oocyte through arising from a distinct cell lineage which the spermathecal constriction into the sperdifferentiate while sperm are maturing matheca (Fig. 3b). The spermatheca (Kimble and Hirsh, 1979). The junction of stretches and appears as if it is being pulled the spermatheca with the oviduct is con- around the oocyte as the oocyte moves. stricted slightly. Oocytes do not pass As the oocyte flows into the spermatheca, through this constriction until they are ma- the lumen of the spermatheca opens and ture. The junction of spermatheca with the sperm are released from the wall of the uterus is a narrow construction of fibrillar spermatheca. These sperm contact the material with a complex morphology (Figs. head end of the advancing oocyte and fer1, 2, and 4). As described below, this con- tilization occurs by the sperm abruptly enstriction is a valve which opens and closes tering into the oocyte as if ingested. It is to allow passage of eggs from spermatheca difficult to observe the actual moment of to the uterus. sperm penetration and we are indebted to We find that hermaphrodite spermatozoa John Sulston for suggesting what to look are morphologically indistinguishable from for. We have recorded three actual penetrathose in the male as described by Wolf et tions on video tape. In all cases the first al. (1978). They are amoeboid cells devoid sperm that contacted the oocyte fertilized of flagella with a pseudopod on one side it, in agreement with J. Sulston’s observaand cell organelles on the other. Sperma- tions (personal communication). Although tozoa accumulate in the central lumen of many other sperm contact the oocytes in the spermatheca oriented with their pseu- the spermatheca, only one sperm penedopods embedded in the walls of the sper- trates, as shown below. Honda (1925) claimed that sperm could penetrate anymatheca (Fig. 2). where on the anterior third of the oocyte, (B) Hermaphrodite Fertilization but our observations are more consistent The process of fertilization has been stud- with a single site of sperm penetration, as ied by direct observation and by analysis of suggested by Nigon et al. (1960). The sperm remains visible inside the oocyte for less video tapes and photomicrographs. Figure than a minute, then disappears amid the 3 shows a sequence of the steps in fertilization observed with the light microscope. yolk granules. Shortly before entering the Oocytes mature and enlarge as they pass spermatheca, the boundary of the oocyte down the oviduct and their nuclei arrest in nucleus becomes less distinct, presumably diakinesis (Nigon, 1949; Hirsh et al., 1976). due to nuclear membrane breakdown and By the time an oocyte reaches the end of reinitiation of meiosis (Figs. 3b and c). Some sperm remain anchored in the the oviduct, it has attained maximum size and become full of yolk granules and its spermatheca as the oocyte squeezes by nucleolus has disappeared. The spermatids them, whereas others appear to be pushed remaining in the oviduct contact the oocyte to the end of the spermatheca by the oocyte
WARD
AND CARREL
Fertilization
and Sperm
Competition
in C. elegans
FIG. 3. Light micrographs of an oocyte during fertilization in an E51 hermaphrodite. Orientation is similar to Fig. 1 but tilted slightly. (a) Mature oocyte being pushed into spermatheca by contraction of the oviduct sheath (open arrows; closed arrows point to sperm). T = 0. (b) Oocyte entering spermatheca and contacting its first sperm (closed arrow). The boundary of the oocyte nucleus (N) has become indistinct. Sperm penetration follows immediately as recorded on video tapes of similar animals but was not captured on this film sequence. T = 1 min. (c) Fertilized oocyte in spermatheca against spermathecal valve SV. Oocyte nucleus (N) is at rear of oocyte. Sperm are noted with closed arrows. 2’ = 3 min. (d) Thin arrows show edges of valve dilating. T = 6 min. (e) Fertilized oocyte in uterus with valve closing behind (arrows). T = 9 min. (f) Fertilized oocyte in uterus. The valve has closed and sperm (arrows) are seen in both the uterus and the spermatheca. Time-lapse analysis shows that the sperm are crawling actively at this stage. T = 20 min. x 650. Bar = 10 pm.
(Figs. 3c and e). On time-lapse video tapes sperm can be seen crawling along the sides of the oocyte as it progresses into the spermatheca.
The fertilized egg normally remains in the spermatheca for 3-10 min. If the hermaphrodite has become sick or damaged, eggs remain longer in the spermatheca and
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DEVELOPMENTAL
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initiate cleavage and embryogenesis there. Ordinarily, the egg pushes up against the spermathecal valve after a few minutes in the spermatheca. This valve dilates to open a passage into the uterus, then the egg slides gently through the valve with little or no distortion (Figs. 3d and e). The valve then closes behind (Figs. 3e and f). Three cases have been observed in which the valve did not open fully but the oocyte squeezed through anyway, in spite of considerable distortion. An example of an oocyte stuck within the valve is shown in Fig. 8c. A clear sign that an oocyte has been fertilized is an increase in Brownian and saltatory motion of the granules in the oocyte cytoplasm. These are presumably yolk and lipid droplets and their increased motility implies a loosening of the cytoplasmic matrix as is observed in other eggs (e.g., Wilson, 1925, Chap. V). This oocyte activation is due to contact with sperm because it does not occur in oocytes passing through the spermatheca of a spermless mutant hermaphrodite (HC17) or a hermaphrodite that has exhausted her sperm supply. Interestingly, the sperm of the fertilizationdefective mutant HCl activate the oocyte granules but do not penetrate or trigger eggshell formation. Eggshell formation appears to begin in the spermatheca, but it is not complete until the oocyte has been in the uterus for about an hour. About 25-30 min after passage into the uterus, the sperm pronucleus becomes visible near the point of sperm penetration. The egg pronucleus then migrates to the sperm pronucleus and fuses and the cleavage commences. The movement of cytoplasm and the pronuclei after fertilization have been described in great detail by Nigon et al. (1960) and by Hirsh et al. (1976). As the fertilized zygote passes through the valve from the spermatheca to the uterus, it carries a number of sperm with it. Time-lapse analysis shows that these sperm
VOLUME
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move actively from the leading end of the zygote back toward the valve and move through the valve to resume positions in the spermatheca. As the valve closes, the movement is particularly active as sperm scramble toward the spermatheca. Figure 4 is an electron micrograph showing sperm extending their pseudopods in the spermathecal valve after passage of a zygote. Once the valve has closed, the sperm regain their positions on the wall of the spermatheta and become less active until the spermatheca opens as the next oocyte enters. (C) Hermaphrodite
Sperm
Utilization
Nigon (1949) observed in C. elegans var. Bergerac that the average progeny per her-
FIG. 4. Electron micrograph showing spermatozoa (S) with pseudopods (P) in the spermathecal valve and in the spermatheca after passage of an oocyte. x 9ooo.
WARD
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Fertilization
and Sperm
Competition
311
in C. elegans
maphrodite was 264 f 67 and the average number of sperm was estimated to be about 370. The experiment summarized in Table 1 shows that in C. elegans var. Bristol the number of sperm corresponds, within experimental error, to the number of progeny throughout the reproductive period. Therefore every sperm is utilized to produce a zygote. For unknown reasons in this particular experiment both the number of sperm and the number of progeny were less than the 280 that we and others (e.g., Hirsh et al., 1976) normally observe. Table 1 also shows that not every oocyte is fertilized by a sperm. The hermaphrodite produces oocytes in excess, as was observed for other C. elegans strains (Nigon, 1965). When most of the sperm are used up, oocytes passing through the spermatheca begin to escape fertilization. These unfertilized oocytes pass down the uterus and are laid together with the zygotes. The shift from fertilized to unfertilized oocytes does not occur abruptly, but begins about 65 hr after hatching at 25°C when the sperm in each spermatheca have dropped below 40. By about 100 hr at 25°C only unfertilized oocytes are laid. The average number laid is about 100, but this is highly variable.
passage into the uterus, the oocyte nucleus undergoes one reductive division even though normal cytoplasmic activation of granules does not occur. After completing this reductive division, the oocyte nucleus returns to the center of the cell and begins chromatin multiplication, becoming manyfold polyploid as determined by Feulgen staining. Direct observation shows that the nuclear membrane breaks down and reforms every 20-30 min, although no karyokinesis occurs. When these unfertilized eggs are laid, they can be distinguished from zygotes under a dissecting microscope because of their brownish color, large nucleus, squashy disklike shape, and uptake of the dye trypan blue. Dye uptake suggests that the egg membranes must be defective and that these are unhealthy cells. The unfertilized eggs that accumulate in fertilization-defective mutants (Ward and Miwa, 1978) appear identical in the light microscope to those in old wild-type hermaphrodites that have exhausted their sperm, except that in fer-1 mutants, at least, cytoplasmic granule activation does occur in oocytes that have contacted defective sperm.
(0) Unfertilized
Adult C. elegans males have a specialized copulatory bursa at their tail and a gonad specialized for continuous sperm production (for description, see Wolf et al., 1978;
“Oocytes”
The unfertilized oocytes do not remain arrested at diakinesis. By observation with Nomarski optics it can be seen that after HERMAPHROTIDE Age (25°C 43 51 64 85
hr)
N
Sperm
5 10 8 8
253 153 44 10
Sperm
(E) Male Fertilization
TABLE 1 SPERM UTILIZATION” used
0 100 209 243
Zygotes 0 94 215 234
Zygotes/sperm
0.94 * 0.15 1.03 f 0.06 0.96 rt_ 0.05
Unfertilized em
2 42
“A synchronized population of hermaphrodites was raised on several plates at 20°C. At the age shown (corrected to 25”C), N worms were fixed, Feulgen stained, and examined to determine the number of sperm remaining and the number used. All sperm were in or near the spermatheca. For the first time point, sperm number was corrected for spermatocytes. Zygotes produced were calculated from progeny and eggs laid plus in utero. All numbers are per worm. The uncertainty shown for zygotes/sperm results from propagating the standard deviations of the mean of the sperm and zygote counts. As the worms aged further, additional unfertilized eggs were laid to an average of lOO/worm.
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DEVELOPMENTAL
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Nelson et al., 1978). When a male worm encounters a hermaphrodite, it reverses and turns to put its bursa in contact with the hermaphrodite. It then moves the bursa over the hermaphrodite’s body until contacting the vulva. Two spicules are inserted into the vulva to anchor the male and open the vagina. Sperm are then ejaculated through the cloaca into the hermaphrodite’s uterus. Figure 5 is a light micrograph of a copulating male showing the sperm being transferred to the hermaphrodite. From live observation of mating and from examination of mated hermaphrodites fixed and stained, it is found that sperm are initially deposited in the uterus near the vulva. These sperm move among the zygotes and spread up the uterus toward the spermatheca. There is substantial variation in the number of sperm transferred to each hermaphrodite on a mating plate or under the compound microscope. The number of copulations varies, the number of ejaculations varies, and many copulations result in no sperm transfer. Even if sperm are transferred, it is commonly observed under the compound microscope that some of them are expelled from the hermaphrodite when the male withdraws his spicules, as shown in Fig. 5.
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73,1979
Time-lapse examination of male sperm transferred to paralyzed hermaphrodites shows that the male sperm begin active crawling within 5 min of transfer. They move over and around the zygotes in the uterus and along the uterine wall. The inset in Fig. 5 shows a male sperm with pseudopods extended. Different sperm move in all directions at once so it is not readily apparent whether or not their motion is directed preferentially toward the spermatheca. The velocities range up to 10 pm/min. Similar sperm movement with a similar velocity has recently been demonstrated in vitro in our laboratory (G. Nelson, unpublished) so it should be possible to analyze the motility in detail and assay for chemotaxis. In several mating experiments an average of 164 male sperm (range O-560) was transferred to hermaphrodites (N = 16) during a 4-hr interval. From direct observation it takes less than 1 hr for these sperm to reach the spermatheca. Not all the sperm deposited by the male reach the spermatheca. Observation of mated hermaphrodites shows that sperm remaining near the vulva are expelled while eggs are being laid. This loss of sperm is minimized to some extent because male copulation temporarily inhibits egg laying (R. Horvitz, personal communication). From the number of progeny produced by hermaphrodites mated in parallel to those used for counting transferred sperm, it appears that all male sperm that reach the spermatheca fertilize an egg. (F) Male Sperm Competition
FIG. 5. Light micrograph of a copulating male just prior to withdrawal of its spicules from the hermaphrodite vulva. Arrows mark sperm in uterus among zygotes (Z) and sperm spilled outside. Inset shows a male sperm with extended pseudopod among zygotes in utero. X 550, inset, X 650.
In some of the male sperm transfer experiments described above, the hermaphrodites mated were young adults that had around 250 of their own sperm at the time of mating. Did male or hermaphrodite sperm fertilize the oocytes? To answer this question the genotype of the zygotes fertilized and laid during intervals following mating was determined. This was done using both wild-type males and hermaphrodites
WARD
AND CARREL
Fertilization
and Sperm
and using several genetically marked strains. Examples of the results of mating hermaphrodites with wild-type males are shown in Fig. 6. This figure shows the fraction of progeny fertilized by male (outcross) sperm as a function of the time after mating for El hermaphrodites. The same result is obtained with wild-type, E224, E364, or El072 hermaphrodites: Within a few hours of mating, many of the mated hermaphrodites produced only outcross progeny. Subsequently some of them continued to produce nothing but outcross progeny, whereas others resumed production of self-progeny. This variation presumably reflects differences in the number of male sperm transferred during the 3-hr mating interval. It is striking that the hermaphrodites invariably produced a maximum proportion of outcross progeny within 20 hr of mating. These results show that male sperm have a competitive advantage at fertilization over the hermaphrodite sperm. We have found that this is true for all male genotypes tested including the rare fertile transformed pseudomales, tra-l(el099), which have two X chromosomes (Hodgkin and Brenner, 1977). Male sperm competition is apparent in Honda’s data, Tables 6 and 7, although he did not recognize it because he did not
m
I
I
0
20
40 TIME
60 IHOURSI
60
100
FIG. 6. Male versus hermaphrodite sperm competition. The fraction of progeny fertilized by N2 male sperm (outcross) is shown as a function of time after mating. Each line is an individual mated El hermaphrodite. Outcross progeny are recognized because they are all nondumpy. A = Nile blue-labeled El467 males. Similar results were obtained with E224, E1072, E364, and wild-type hermaphrodites. M = interval of mating.
Competition
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know the mechanism of sex determination (Honda, 1925). Sperm competition has been observed in a number of dioecious insect species in which females can be inseminated by more than one male (reviewed by Parker (1970) and more recently discussed by Prout and Bundgaard (1977)). In most cases, the second male’s sperm have a competitive advantage, but it is rarely as total an advantage as seen between C. elegans male and hermaphrodite sperm. We have attempted to distinguish among several possible mechanisms for competition. First, the male sperm might be transferred in numbers which overwhelm the hermaphrodite’s fixed number of sperm. The quantitation of sperm transfer described above shows that this is not the case. This is also supported by the observation that some mated hermaphrodites only produce a few outcross progeny (Fig. 6) but they still produce them shortly after mating. Second, the male sperm might somehow contact the oocytes because they arrive in the spermatheca after the hermaphrodite sperm. Since fertilization occurs near the oviduct, and the male sperm enter from the uterus, this seems unlikely. Nonetheless, the sperm might arrange themselves in the spermatheca in such a way that the last sperm in are the first sperm utilized. This is the most common mechanism of insect sperm competition (Parker, 1970). To test this possibility we examined sperm competition between the sperm of two males mated successively to a hermaphrodite. This experiment was done using several different combinations of genetic markers to distinguish each male’s progeny. To be sure that the hermaphrodite spermatheca would have room for sperm of both males, hermaphrodites with a temperature-sensitive mutation that blocks their own spermatogenesis, isx-l(hcl7ts), were used for most experiments, although the same result was obtained with fertile hermaphrodites.
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The spermless hermaphrodite is a “true” female so this experiment is similar to studying sperm competition in a dioecious organism. The results are summarized in Fig. 7, which plots the fraction of second male progeny as a function of time, analogous to Fig. 6. The results are different from the male versus hermaphrodite case. The second male sperm do not appear to have a competitive advantage over the first. The fraction of second male progeny increases and levels off gradually in most cases, as if the sperm were gradually mixing. In two cases, no second male progeny were produced until 20 hr after mating, whereas this was never seen in male versus hermaphrodite sperm competition. This result shows that sperm are not arranged and utilized in the spermatheca in the order of entry. It also shows that the competitive advantage of male sperm is only over hermaphrodite sperm and not over other male sperm.
(G) The Arrangement of Male Sperm in a Mated Hermaphrodite In order to determine how the male sperm were arranged in the spermatheca after mating, a way of distinguishing male from hermaphrodite sperm was sought. It
2 .6 z .4 0 t,g.8iG, .2 M'M2 2 Lo 0
‘\ \ \&---A--
,
,
,p-----'
20
40 TIME
60 ( HOURS
80
100
1
FIG. 7. Male versus male sperm competition. HCD4 hermaphrodites raised at 25°C so that they would have no sperm of their own were used for most experiments Ml and M2 show the duration of the first and second male mating, respectively. Solid lines show N2 males followed by E364 males. Dashed lines show E364 males followed by N2 males. Several other combinations of male and hermaphrodite genotypes gave similar results, as did mating by Nile blue A-labeled males.
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was found that the lipid stain, Nile blue A, could be used as a fluorescent vital dye which would be taken up by males and incorporated into all their tissues, including the sperm. Single sperm were readily visualized by fluorescence after extrusion from a male and remained fluorescent for several hours. Labeled males were fertile and their sperm were used preferentially in competition with hermaphrodite sperm (Fig. 6), but not used preferentially in competition with male sperm (data not shown). It was found that shortly after mating, the fluorescent sperm distribute among the eggs and move up the uterus as expected. Shortly after the sperm reach the spermatheta, they appear to accumulate preferentially against the wall of the spermatheca with unlabeled hermaphrodite sperm located in the center (Figs. 8a, b). Very quickly the fluorescence is transferred from the sperm to the walls of the spermatheca, so that a few minutes after sperm enter the spermatheca only the spermatheca is labeled (Figs. SC, d). Such dye transfer is common to lipid-soluble dyes when membranes come in contact (e.g., Lentz et al., 1976) so the transfer of dye supports the observation that the male sperm are in close contact with the walls of the spermatheta. No dye transfer was observed to eggs or the walls of the uterus, and in the squashed hermaphrodites only the remnants of the spermatheca were fluorescent. These results suggest that the male sperm take up positions anchored to the wall of the spermatheca, displacing hermaphrodite sperm to the central lumen. This displacement could explain the preferential sperm utilization if sperm anchored to the spermathecal wall do not get swept down the spermatheca and thus reach OOcytes first. It is not clear whether this displacement is the only mechanism to account for preferential male sperm utilization. The male sperm might have a higher affinity for oocytes or might release an inhibitory factor that somehow inactivates
WARD
AND CARREL
Fertilization
and Sperm
Competition
in C. elegans
FIG. 8. Nile blue A-labeled male sperm transfer to an E51 hermaphrodite. (a, c) Nomarski optics; (b, d) fluorescence. (a, b) Spermatheca immediately after male sperm entered. Note stronger fluorescence from sperm on the periphery against the wall of the spermatheca. Some fluorescence is contributed by sperm above and below the plane of focus. (c, d) Oocyte arrested in spermathecal valve more than 1 hr after mating. Only the walls of the spermatheca are fluorescent. Dissection of this and similar mated hermaphrodites after live observation confirmed that only the walls of the spermatheca were fluorescent; no fluorescence was retained by spermatozoa.
the hermaphrodite sperm. Support for this latter possibility is presented in Section I. (H) Stimulation
of Oogenesis
by Mating
A second consequence of mating by males is observed when the total number of oocytes (fertilized plus unfertilized) produced by mated and unmated hermaphrodites is compared. Table 2 shows the results of mating El hermaphrodites. The column labeled total oocytes/worm shows that those mated hermaphrodites that produced mostly outcross progeny yielded significantly more total oocytes than unmated controls. Similar results have been obtained with wild-type and E224 hermaphrodites. These observations imply that oogenesis or oocyte maturation is stimulated by mating.
Analysis of the rate of egg laying by mated hermaphrodites showed that the increase in progeny production is due to an increase in the duration of production of progeny rather than an increase in the rate. It was also observed that old wild-type hermaphrodites that have ceased laying oocytes can be stimulated to continue oocyte production by mating. Several temperature-sensitive mutants have been isolated that lay very few oocytes at restrictive temperature. Many of these can be stimulated to produce large numbers of progeny if mated by males (Hirsh and Vanderslice, 1976; Nelson et al., 1978; D. Hirsh and L. Edgar, personal communication; J. Miwa and S. Ward, unpublished observations). At least one of these mutants
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VOLUME
TABLE THE
EFFECT
OF MATING
N Unmated control Mated, >0.8 outcross Mated, no outcross
10 5 17
2
ON PROGENY
Progeny/worm 199 + 10 479 f 84 163 -t 24
73,1979
AND OOCYTE P
BIOLOGY
Fertilization
73,304-321
(1979)
and Sperm Competition in the Nematode elegans
Caenorhabditis
SAMUEL WARD AND JOHN S. CARREL Carnegie Maryland
Institution of Washington, Department 21210, and Department of Biological
Received
December
of Embryology, 115 West University Chemistry, Harvard Medical School, 02115
4, 1978; accepted
in revised
form
June
Parkway, Baltimore, Boston, Massachusetts
6, 1979
The process of fertilization by hermaphrodite and male sperm is described. In the hermaphrodite fertilization occurs in the spermatheca by the first sperm to contact the oocyte. Other sperm that contact the oocyte are swept into the uterus but they crawl back into the spermatheca to fertilize subsequent oocytes so that every sperm fertilizes an oocyte. Not every oocyte is fertilized because oocytes are made in excess. Fertilization triggers active movement of oocyte cytoplasmic granules. Sperm penetration is not required for this activation because fertilization-defective mutant sperm trigger activation without penetration. When males copulate with hermaphrodites, their sperm is deposited in the uterus beneath the vulva. These sperm crawl up the uterus to the spermatheca where they displace the hermaphrodite sperm from the spermathecal walls and preferentially fertilize the oocytes. This preferential fertilization appears to be due in part to inhibition of hermaphrodite sperm fertility by the male sperm. The male sperm competition ensures male sperm utilization and thus some outcrossing in a population of predominantly self-fertilizating hermaphrodites. INTRODUCTION
cundity of the hermaphrodite was limited by its production of sperm and that oocytes were produced in excess. Maupas also observed that males were produced in the population at a frequency of about 0.001 but the males he studied were invariably sterile. Maupas’ observations were repeated on a very similar species by Potts (1910), and again on C. elegans isolated near Philadelphia by Honda (1925). Honda was the first worker to obtain males that were fertile. Nigon (1949), in a classic comparative study of nematode cytology and reproduction, established the chromosomal basis of sex determination (hermaphrodites are 5AA,XX; males are 5AA,X) and confirmed the studies of the earlier workers on yet another C. elegans isolate, this one from Bergerac in France. This work was reviewed by Nigon (1965). The work presented here extends these previous studies. By examination of live specimens with light microscopy and fixed
Many specialized gene products must participate in the specific interactions of gametes during fertilization. Some of these gene products will determine the surface topology of the gametes and the specificity of their interaction; others will be required for sperm motility and penetration. In order to identify some of these products and to specify their function, we have begun to isolate and characterize fertilization-defective mutants in the nematode Caenorhabditis elegans (Ward, 1977a; Ward and Miwa, 1978). Before mutants can be studied profitably, it is necessary to understand the normal process of fertilization. A number of studies of C. elegans reproduction have been reported since its isolation some 80 years ago. Maupas (1900) first isolated C. elegans (then called Rhabditis eZegans) in Algeria and described its hermaphroditic mode of reproduction. He discovered that the fe304 0012-1606/79/120304-18$02.00/O Copyright All rights
0 1979 by Academic Press, Inc. of reproduction in any form reserved.
WARD
AND
CARREL
Fertilization
specimens with electron microscopy, we have examined the process of fertilization in more detail than before. We describe the movements of the sperm and the activation of the egg. We also report the phenomenon of male sperm competition, analyze its mechanism, and interpret its significance. These studies provide a detailed background for interpretation of fertilization-defective mutants. MATERIALS
AND
METHODS
Strains and worm culture. Wild-type strain N2 of Caenorhabditis elegans var Bristol was obtained from S. Brenner in 1973. The following mutant strains were E224=dpyemployed: El=dpy-l(e1); ll(e224); E369=unc-51(e369); E51=unc13(e51); E1072=unc-29(e1072); E1035=chel(elO35)fer-l(hc1); HC17=isx-l(hcl7ts); EH66=tra-l(el099)/dpy-18(e364); HCl=ferl(hclts); HC-D4=isx-l(hcl7)dpy-18(e364); E1467=him-l(e1467). E strains are from Brenner’s collection, HC strains from our laboratory. Strains were maintained at 16 or 20°C on NGM plates seeded with E. coli strain OP50 as described by Brenner (1974). Synchronized populations were prepared when needed by collecting worms hatched during a 1-hr interval. Mating experiments. All matings were on 6-cm petri plates of NGM agar seeded at the center with a tiny spot of E. coli 12 hr before mating. Males for mating were picked as juveniles and maintained as virgins for 24 to 48 hr. Ten to 15 males were added with 4 hermaphrodites to each mating plate. For sperm competition experiments hermaphrodites that had laid l-10 zygotes prior to mating were used. After mating for 3-7 hr individual hermaphrodites were transferred to fresh plates at 12to 24-hr intervals and the genotypes of their progeny scored after they had matured. Vital staining. A stock solution of Nile blue A (Allied Chemical) (5% in 95% ethanol) was diluted &fold with M-9 buffer (Brenner, 1974) and 6 to 8 drops were added
and Sperm
Competition
in C. elegans
305
to a 6-cm NGM plate preseeded with E. coli. Males were labeled by placing El467 larvae on a Nile blue A plate. When males had begun to mature they were transferred to a fresh Nile blue A plate for 24-36 hr prior to mating. At the concentration used, the dye does not prevent worm growth or reproduction. Light microscopy. All observations and photographs were made with a Zeiss Universal microscope equipped for Nomarski differential interference contrast observation and epifluorescence. Nile blue A fluorescence was observed with a Zeiss filter combination 486615 which excites with green light and passes red fluorescence for visualization. Nomarski images were recorded on Kodak SO-115 film developed in HCllO dilution D. Fluorescence images were recorded on Kodak Recording Film 2475 developed according to instructions. Identical fields were recorded by interchanging camera backs without altering focus using Planapochromatic lenses to ensure that the focal plane did not change in going from the white light of Nomarski to the red of fluorescence. For observation of live specimens, worms were mounted on slabs of agar under a coverslip as described by Sulston (1976). Uncoordinated worms were usually used, especially the paralyzed E369 because they remain still for time-lapse observation. Photographs were taken with a Zeiss microflash. Detailed studies of fertilization were made from video tapes recorded on a Panasonic NV8030 time-lapse video tape recorder using a Panasonic Nevicon camera. Sperm motility and egg granule activation were recorded at l/18 speed and examined at l/l. Stained preparations were prepared and examined as described previously (Ward and Miwa, 1978). Electron microscopy. Worms were fixed in 1% 0~0~ in 0.08 sodium phosphate (pH 7.4) or 3% glutaraldehyde followed by 0~0~ and prepared for sectioning as described by
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Ward et al. (1975), except that they were cut near the spermatheca in the first fixative to ensure penetration. Sections were examined at 60 or 80 kV with a Zeiss EM10 or JEOL 100s electron microscope. RESULTS
(A) Hermaphrodite
Sperm Development
Light microscopic studies of the development of C. elegans reproductive system were presented by Honda (1925) and again by Nigon (1949, 1965). Hirsh et al. (1976) have described oogenesis with electron microscopy, and Kimble and Hirsh (1979) have described the complete development of the somatic gonad structures. Male sperm development has been described
Oviduct
VOLUME 73,1979
with electron microscopy by Wolf et al. (1978). Hermaphrodite spermatogenesis begins around 24 hr after hatching at 25°C. Primary spermatocytes enter prophase I in the proximal arm of the gonad prior to the gonad’s 180’ turn. By about 35 hr the first spermatocytes have reached diakinesis. The first haploid nuclei become visible at about 39 hr, which corresponds to the time of the last moult. Sperm in the anterior arm of the gonad often mature about 1 hr ahead of those in the posterior arm. Spermiogenesis (the maturation of haploid spermatids) proceeds simultaneously with spermatogenesis from 39 until 48-50 hr. In many adult hermaphrodites fixed and
Spe rmat
heca
Uterus
FIG. I. (a and b) Light micrographs of adult hermaphrodite gonads showing the region of the spermatheca. Orientation is as shown diagrammatically in (c), which shows round spermatids against the oocyte in the oviduct and mature spermatozoa against the wall of the spermatheca. The distal arm of the gonad with oogonia is below in (a) and to the lower left in (b). (a) An older adult with a nearly mature oocyte in the oviduct. x 650. (b) A younger adult showing spermatids in the oviduct and mature spermatozoa in the spermatheca. x 1000. Arrows indicate spermatozoa. Sd = spermatid; 00 = oocyte; Zyg = zygote; SV = spermathecal valve joining the uterus; N = nucleus. Bar = 10 pm.
WARD
AND
CARREL
Fertilization
stained from 50 to 150 hr after hatching, we observed no new spermatocytes or spermatids, so sperm development occurs only once as the earlier workers showed. Spermatozoa first appear in the ovotestis among the spermatocytes and spermatids. As observed in other nematodes (e.g., Chitwood and Chitwood, 1974, p. 192), the spermatids are round cells that look grainy in the light microscope. As they complete maturation to spermatozoa they become irregular and less grainy (Fig. lb). With the electron microscope it can be seen that this alteration is due to fusion of sperm organ-
and Sperm
Competition
in C. elegans
307
elles called membranous organelles (or special membrane structures) with the plasma membrane and extension of a pseudopod (Fig. 2). This maturation is seen in some males (Wolf et al., 1978) and has been described in several other nematodes (e.g., Foor, 1970; Burghardt and Foor, 1978; McLaren, 1973). Passage of the first mature oocyte pushes sperm from the ovotestis into the spermatheta. Our observations suggest that some sperm crawl in as well. Only mature spermatozoa are found in the spermatheca, and after several oocytes have passed through,
FIG. 2. Electron micrograph of a longitudinal section of the spermatheca corresponding to the view shown in Fig. la. 0~0, fixation. Mature spermatozoa (S) have an electron-opaque nucleus, an electron-dense cytoplasm around the nucleus, and an irregular pseudopod (I?). They are embedded in the wall of the spermatheca. The one spermatid shown in the oviduct in this section has no pseudopod and unfused membranous organelles. In many other sectioned adult hermaphrodites, similar spermatids and no spermatozoa are seen in the oviduct, The nematode’s gut is above the gonad and a zygote in the uterus to the right is badly fixed because of its impermeable eggshell. x 7000.
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only immature spermatids are found in the there, but they do not fertilize. The oocyte ovotestis (now the oviduct) (Fig. 2 and its completes its maturation as its nucleus milegend). It may be that only mature sper- grates from the center of the cell to the end matozoa are carried into the spermatheca distal from the spermatheca (Figs. la and or that spermatids carried into the sper- 3a). This migration is accompanied by spomatheca rapidly complete maturation radic contractions of the oviduct walls and there. contractions of regions of oocyte cytoplasm. The spermatheca is a thick-walled spiral Contractions of the oviduct wall increase tube between the oviduct and the uterus (Figs. 3a and b) and shortly thereafter these (Figs. 1 and 2). It is formed of 24 cells contractions squeeze the oocyte through arising from a distinct cell lineage which the spermathecal constriction into the sperdifferentiate while sperm are maturing matheca (Fig. 3b). The spermatheca (Kimble and Hirsh, 1979). The junction of stretches and appears as if it is being pulled the spermatheca with the oviduct is con- around the oocyte as the oocyte moves. stricted slightly. Oocytes do not pass As the oocyte flows into the spermatheca, through this constriction until they are ma- the lumen of the spermatheca opens and ture. The junction of spermatheca with the sperm are released from the wall of the uterus is a narrow construction of fibrillar spermatheca. These sperm contact the material with a complex morphology (Figs. head end of the advancing oocyte and fer1, 2, and 4). As described below, this con- tilization occurs by the sperm abruptly enstriction is a valve which opens and closes tering into the oocyte as if ingested. It is to allow passage of eggs from spermatheca difficult to observe the actual moment of to the uterus. sperm penetration and we are indebted to We find that hermaphrodite spermatozoa John Sulston for suggesting what to look are morphologically indistinguishable from for. We have recorded three actual penetrathose in the male as described by Wolf et tions on video tape. In all cases the first al. (1978). They are amoeboid cells devoid sperm that contacted the oocyte fertilized of flagella with a pseudopod on one side it, in agreement with J. Sulston’s observaand cell organelles on the other. Sperma- tions (personal communication). Although tozoa accumulate in the central lumen of many other sperm contact the oocytes in the spermatheca oriented with their pseu- the spermatheca, only one sperm penedopods embedded in the walls of the sper- trates, as shown below. Honda (1925) claimed that sperm could penetrate anymatheca (Fig. 2). where on the anterior third of the oocyte, (B) Hermaphrodite Fertilization but our observations are more consistent The process of fertilization has been stud- with a single site of sperm penetration, as ied by direct observation and by analysis of suggested by Nigon et al. (1960). The sperm remains visible inside the oocyte for less video tapes and photomicrographs. Figure than a minute, then disappears amid the 3 shows a sequence of the steps in fertilization observed with the light microscope. yolk granules. Shortly before entering the Oocytes mature and enlarge as they pass spermatheca, the boundary of the oocyte down the oviduct and their nuclei arrest in nucleus becomes less distinct, presumably diakinesis (Nigon, 1949; Hirsh et al., 1976). due to nuclear membrane breakdown and By the time an oocyte reaches the end of reinitiation of meiosis (Figs. 3b and c). Some sperm remain anchored in the the oviduct, it has attained maximum size and become full of yolk granules and its spermatheca as the oocyte squeezes by nucleolus has disappeared. The spermatids them, whereas others appear to be pushed remaining in the oviduct contact the oocyte to the end of the spermatheca by the oocyte
WARD
AND CARREL
Fertilization
and Sperm
Competition
in C. elegans
FIG. 3. Light micrographs of an oocyte during fertilization in an E51 hermaphrodite. Orientation is similar to Fig. 1 but tilted slightly. (a) Mature oocyte being pushed into spermatheca by contraction of the oviduct sheath (open arrows; closed arrows point to sperm). T = 0. (b) Oocyte entering spermatheca and contacting its first sperm (closed arrow). The boundary of the oocyte nucleus (N) has become indistinct. Sperm penetration follows immediately as recorded on video tapes of similar animals but was not captured on this film sequence. T = 1 min. (c) Fertilized oocyte in spermatheca against spermathecal valve SV. Oocyte nucleus (N) is at rear of oocyte. Sperm are noted with closed arrows. 2’ = 3 min. (d) Thin arrows show edges of valve dilating. T = 6 min. (e) Fertilized oocyte in uterus with valve closing behind (arrows). T = 9 min. (f) Fertilized oocyte in uterus. The valve has closed and sperm (arrows) are seen in both the uterus and the spermatheca. Time-lapse analysis shows that the sperm are crawling actively at this stage. T = 20 min. x 650. Bar = 10 pm.
(Figs. 3c and e). On time-lapse video tapes sperm can be seen crawling along the sides of the oocyte as it progresses into the spermatheca.
The fertilized egg normally remains in the spermatheca for 3-10 min. If the hermaphrodite has become sick or damaged, eggs remain longer in the spermatheca and
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DEVELOPMENTAL
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initiate cleavage and embryogenesis there. Ordinarily, the egg pushes up against the spermathecal valve after a few minutes in the spermatheca. This valve dilates to open a passage into the uterus, then the egg slides gently through the valve with little or no distortion (Figs. 3d and e). The valve then closes behind (Figs. 3e and f). Three cases have been observed in which the valve did not open fully but the oocyte squeezed through anyway, in spite of considerable distortion. An example of an oocyte stuck within the valve is shown in Fig. 8c. A clear sign that an oocyte has been fertilized is an increase in Brownian and saltatory motion of the granules in the oocyte cytoplasm. These are presumably yolk and lipid droplets and their increased motility implies a loosening of the cytoplasmic matrix as is observed in other eggs (e.g., Wilson, 1925, Chap. V). This oocyte activation is due to contact with sperm because it does not occur in oocytes passing through the spermatheca of a spermless mutant hermaphrodite (HC17) or a hermaphrodite that has exhausted her sperm supply. Interestingly, the sperm of the fertilizationdefective mutant HCl activate the oocyte granules but do not penetrate or trigger eggshell formation. Eggshell formation appears to begin in the spermatheca, but it is not complete until the oocyte has been in the uterus for about an hour. About 25-30 min after passage into the uterus, the sperm pronucleus becomes visible near the point of sperm penetration. The egg pronucleus then migrates to the sperm pronucleus and fuses and the cleavage commences. The movement of cytoplasm and the pronuclei after fertilization have been described in great detail by Nigon et al. (1960) and by Hirsh et al. (1976). As the fertilized zygote passes through the valve from the spermatheca to the uterus, it carries a number of sperm with it. Time-lapse analysis shows that these sperm
VOLUME
73.1979
move actively from the leading end of the zygote back toward the valve and move through the valve to resume positions in the spermatheca. As the valve closes, the movement is particularly active as sperm scramble toward the spermatheca. Figure 4 is an electron micrograph showing sperm extending their pseudopods in the spermathecal valve after passage of a zygote. Once the valve has closed, the sperm regain their positions on the wall of the spermatheta and become less active until the spermatheca opens as the next oocyte enters. (C) Hermaphrodite
Sperm
Utilization
Nigon (1949) observed in C. elegans var. Bergerac that the average progeny per her-
FIG. 4. Electron micrograph showing spermatozoa (S) with pseudopods (P) in the spermathecal valve and in the spermatheca after passage of an oocyte. x 9ooo.
WARD
AND CARREL
Fertilization
and Sperm
Competition
311
in C. elegans
maphrodite was 264 f 67 and the average number of sperm was estimated to be about 370. The experiment summarized in Table 1 shows that in C. elegans var. Bristol the number of sperm corresponds, within experimental error, to the number of progeny throughout the reproductive period. Therefore every sperm is utilized to produce a zygote. For unknown reasons in this particular experiment both the number of sperm and the number of progeny were less than the 280 that we and others (e.g., Hirsh et al., 1976) normally observe. Table 1 also shows that not every oocyte is fertilized by a sperm. The hermaphrodite produces oocytes in excess, as was observed for other C. elegans strains (Nigon, 1965). When most of the sperm are used up, oocytes passing through the spermatheca begin to escape fertilization. These unfertilized oocytes pass down the uterus and are laid together with the zygotes. The shift from fertilized to unfertilized oocytes does not occur abruptly, but begins about 65 hr after hatching at 25°C when the sperm in each spermatheca have dropped below 40. By about 100 hr at 25°C only unfertilized oocytes are laid. The average number laid is about 100, but this is highly variable.
passage into the uterus, the oocyte nucleus undergoes one reductive division even though normal cytoplasmic activation of granules does not occur. After completing this reductive division, the oocyte nucleus returns to the center of the cell and begins chromatin multiplication, becoming manyfold polyploid as determined by Feulgen staining. Direct observation shows that the nuclear membrane breaks down and reforms every 20-30 min, although no karyokinesis occurs. When these unfertilized eggs are laid, they can be distinguished from zygotes under a dissecting microscope because of their brownish color, large nucleus, squashy disklike shape, and uptake of the dye trypan blue. Dye uptake suggests that the egg membranes must be defective and that these are unhealthy cells. The unfertilized eggs that accumulate in fertilization-defective mutants (Ward and Miwa, 1978) appear identical in the light microscope to those in old wild-type hermaphrodites that have exhausted their sperm, except that in fer-1 mutants, at least, cytoplasmic granule activation does occur in oocytes that have contacted defective sperm.
(0) Unfertilized
Adult C. elegans males have a specialized copulatory bursa at their tail and a gonad specialized for continuous sperm production (for description, see Wolf et al., 1978;
“Oocytes”
The unfertilized oocytes do not remain arrested at diakinesis. By observation with Nomarski optics it can be seen that after HERMAPHROTIDE Age (25°C 43 51 64 85
hr)
N
Sperm
5 10 8 8
253 153 44 10
Sperm
(E) Male Fertilization
TABLE 1 SPERM UTILIZATION” used
0 100 209 243
Zygotes 0 94 215 234
Zygotes/sperm
0.94 * 0.15 1.03 f 0.06 0.96 rt_ 0.05
Unfertilized em
2 42
“A synchronized population of hermaphrodites was raised on several plates at 20°C. At the age shown (corrected to 25”C), N worms were fixed, Feulgen stained, and examined to determine the number of sperm remaining and the number used. All sperm were in or near the spermatheca. For the first time point, sperm number was corrected for spermatocytes. Zygotes produced were calculated from progeny and eggs laid plus in utero. All numbers are per worm. The uncertainty shown for zygotes/sperm results from propagating the standard deviations of the mean of the sperm and zygote counts. As the worms aged further, additional unfertilized eggs were laid to an average of lOO/worm.
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DEVELOPMENTAL
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Nelson et al., 1978). When a male worm encounters a hermaphrodite, it reverses and turns to put its bursa in contact with the hermaphrodite. It then moves the bursa over the hermaphrodite’s body until contacting the vulva. Two spicules are inserted into the vulva to anchor the male and open the vagina. Sperm are then ejaculated through the cloaca into the hermaphrodite’s uterus. Figure 5 is a light micrograph of a copulating male showing the sperm being transferred to the hermaphrodite. From live observation of mating and from examination of mated hermaphrodites fixed and stained, it is found that sperm are initially deposited in the uterus near the vulva. These sperm move among the zygotes and spread up the uterus toward the spermatheca. There is substantial variation in the number of sperm transferred to each hermaphrodite on a mating plate or under the compound microscope. The number of copulations varies, the number of ejaculations varies, and many copulations result in no sperm transfer. Even if sperm are transferred, it is commonly observed under the compound microscope that some of them are expelled from the hermaphrodite when the male withdraws his spicules, as shown in Fig. 5.
VOLUME
73,1979
Time-lapse examination of male sperm transferred to paralyzed hermaphrodites shows that the male sperm begin active crawling within 5 min of transfer. They move over and around the zygotes in the uterus and along the uterine wall. The inset in Fig. 5 shows a male sperm with pseudopods extended. Different sperm move in all directions at once so it is not readily apparent whether or not their motion is directed preferentially toward the spermatheca. The velocities range up to 10 pm/min. Similar sperm movement with a similar velocity has recently been demonstrated in vitro in our laboratory (G. Nelson, unpublished) so it should be possible to analyze the motility in detail and assay for chemotaxis. In several mating experiments an average of 164 male sperm (range O-560) was transferred to hermaphrodites (N = 16) during a 4-hr interval. From direct observation it takes less than 1 hr for these sperm to reach the spermatheca. Not all the sperm deposited by the male reach the spermatheca. Observation of mated hermaphrodites shows that sperm remaining near the vulva are expelled while eggs are being laid. This loss of sperm is minimized to some extent because male copulation temporarily inhibits egg laying (R. Horvitz, personal communication). From the number of progeny produced by hermaphrodites mated in parallel to those used for counting transferred sperm, it appears that all male sperm that reach the spermatheca fertilize an egg. (F) Male Sperm Competition
FIG. 5. Light micrograph of a copulating male just prior to withdrawal of its spicules from the hermaphrodite vulva. Arrows mark sperm in uterus among zygotes (Z) and sperm spilled outside. Inset shows a male sperm with extended pseudopod among zygotes in utero. X 550, inset, X 650.
In some of the male sperm transfer experiments described above, the hermaphrodites mated were young adults that had around 250 of their own sperm at the time of mating. Did male or hermaphrodite sperm fertilize the oocytes? To answer this question the genotype of the zygotes fertilized and laid during intervals following mating was determined. This was done using both wild-type males and hermaphrodites
WARD
AND CARREL
Fertilization
and Sperm
and using several genetically marked strains. Examples of the results of mating hermaphrodites with wild-type males are shown in Fig. 6. This figure shows the fraction of progeny fertilized by male (outcross) sperm as a function of the time after mating for El hermaphrodites. The same result is obtained with wild-type, E224, E364, or El072 hermaphrodites: Within a few hours of mating, many of the mated hermaphrodites produced only outcross progeny. Subsequently some of them continued to produce nothing but outcross progeny, whereas others resumed production of self-progeny. This variation presumably reflects differences in the number of male sperm transferred during the 3-hr mating interval. It is striking that the hermaphrodites invariably produced a maximum proportion of outcross progeny within 20 hr of mating. These results show that male sperm have a competitive advantage at fertilization over the hermaphrodite sperm. We have found that this is true for all male genotypes tested including the rare fertile transformed pseudomales, tra-l(el099), which have two X chromosomes (Hodgkin and Brenner, 1977). Male sperm competition is apparent in Honda’s data, Tables 6 and 7, although he did not recognize it because he did not
m
I
I
0
20
40 TIME
60 IHOURSI
60
100
FIG. 6. Male versus hermaphrodite sperm competition. The fraction of progeny fertilized by N2 male sperm (outcross) is shown as a function of time after mating. Each line is an individual mated El hermaphrodite. Outcross progeny are recognized because they are all nondumpy. A = Nile blue-labeled El467 males. Similar results were obtained with E224, E1072, E364, and wild-type hermaphrodites. M = interval of mating.
Competition
in C. elegans
313
know the mechanism of sex determination (Honda, 1925). Sperm competition has been observed in a number of dioecious insect species in which females can be inseminated by more than one male (reviewed by Parker (1970) and more recently discussed by Prout and Bundgaard (1977)). In most cases, the second male’s sperm have a competitive advantage, but it is rarely as total an advantage as seen between C. elegans male and hermaphrodite sperm. We have attempted to distinguish among several possible mechanisms for competition. First, the male sperm might be transferred in numbers which overwhelm the hermaphrodite’s fixed number of sperm. The quantitation of sperm transfer described above shows that this is not the case. This is also supported by the observation that some mated hermaphrodites only produce a few outcross progeny (Fig. 6) but they still produce them shortly after mating. Second, the male sperm might somehow contact the oocytes because they arrive in the spermatheca after the hermaphrodite sperm. Since fertilization occurs near the oviduct, and the male sperm enter from the uterus, this seems unlikely. Nonetheless, the sperm might arrange themselves in the spermatheca in such a way that the last sperm in are the first sperm utilized. This is the most common mechanism of insect sperm competition (Parker, 1970). To test this possibility we examined sperm competition between the sperm of two males mated successively to a hermaphrodite. This experiment was done using several different combinations of genetic markers to distinguish each male’s progeny. To be sure that the hermaphrodite spermatheca would have room for sperm of both males, hermaphrodites with a temperature-sensitive mutation that blocks their own spermatogenesis, isx-l(hcl7ts), were used for most experiments, although the same result was obtained with fertile hermaphrodites.
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DEVELOPMENTAL
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The spermless hermaphrodite is a “true” female so this experiment is similar to studying sperm competition in a dioecious organism. The results are summarized in Fig. 7, which plots the fraction of second male progeny as a function of time, analogous to Fig. 6. The results are different from the male versus hermaphrodite case. The second male sperm do not appear to have a competitive advantage over the first. The fraction of second male progeny increases and levels off gradually in most cases, as if the sperm were gradually mixing. In two cases, no second male progeny were produced until 20 hr after mating, whereas this was never seen in male versus hermaphrodite sperm competition. This result shows that sperm are not arranged and utilized in the spermatheca in the order of entry. It also shows that the competitive advantage of male sperm is only over hermaphrodite sperm and not over other male sperm.
(G) The Arrangement of Male Sperm in a Mated Hermaphrodite In order to determine how the male sperm were arranged in the spermatheca after mating, a way of distinguishing male from hermaphrodite sperm was sought. It
2 .6 z .4 0 t,g.8iG, .2 M'M2 2 Lo 0
‘\ \ \&---A--
,
,
,p-----'
20
40 TIME
60 ( HOURS
80
100
1
FIG. 7. Male versus male sperm competition. HCD4 hermaphrodites raised at 25°C so that they would have no sperm of their own were used for most experiments Ml and M2 show the duration of the first and second male mating, respectively. Solid lines show N2 males followed by E364 males. Dashed lines show E364 males followed by N2 males. Several other combinations of male and hermaphrodite genotypes gave similar results, as did mating by Nile blue A-labeled males.
VOLUME
73,1979
was found that the lipid stain, Nile blue A, could be used as a fluorescent vital dye which would be taken up by males and incorporated into all their tissues, including the sperm. Single sperm were readily visualized by fluorescence after extrusion from a male and remained fluorescent for several hours. Labeled males were fertile and their sperm were used preferentially in competition with hermaphrodite sperm (Fig. 6), but not used preferentially in competition with male sperm (data not shown). It was found that shortly after mating, the fluorescent sperm distribute among the eggs and move up the uterus as expected. Shortly after the sperm reach the spermatheta, they appear to accumulate preferentially against the wall of the spermatheca with unlabeled hermaphrodite sperm located in the center (Figs. 8a, b). Very quickly the fluorescence is transferred from the sperm to the walls of the spermatheca, so that a few minutes after sperm enter the spermatheca only the spermatheca is labeled (Figs. SC, d). Such dye transfer is common to lipid-soluble dyes when membranes come in contact (e.g., Lentz et al., 1976) so the transfer of dye supports the observation that the male sperm are in close contact with the walls of the spermatheta. No dye transfer was observed to eggs or the walls of the uterus, and in the squashed hermaphrodites only the remnants of the spermatheca were fluorescent. These results suggest that the male sperm take up positions anchored to the wall of the spermatheca, displacing hermaphrodite sperm to the central lumen. This displacement could explain the preferential sperm utilization if sperm anchored to the spermathecal wall do not get swept down the spermatheca and thus reach OOcytes first. It is not clear whether this displacement is the only mechanism to account for preferential male sperm utilization. The male sperm might have a higher affinity for oocytes or might release an inhibitory factor that somehow inactivates
WARD
AND CARREL
Fertilization
and Sperm
Competition
in C. elegans
FIG. 8. Nile blue A-labeled male sperm transfer to an E51 hermaphrodite. (a, c) Nomarski optics; (b, d) fluorescence. (a, b) Spermatheca immediately after male sperm entered. Note stronger fluorescence from sperm on the periphery against the wall of the spermatheca. Some fluorescence is contributed by sperm above and below the plane of focus. (c, d) Oocyte arrested in spermathecal valve more than 1 hr after mating. Only the walls of the spermatheca are fluorescent. Dissection of this and similar mated hermaphrodites after live observation confirmed that only the walls of the spermatheca were fluorescent; no fluorescence was retained by spermatozoa.
the hermaphrodite sperm. Support for this latter possibility is presented in Section I. (H) Stimulation
of Oogenesis
by Mating
A second consequence of mating by males is observed when the total number of oocytes (fertilized plus unfertilized) produced by mated and unmated hermaphrodites is compared. Table 2 shows the results of mating El hermaphrodites. The column labeled total oocytes/worm shows that those mated hermaphrodites that produced mostly outcross progeny yielded significantly more total oocytes than unmated controls. Similar results have been obtained with wild-type and E224 hermaphrodites. These observations imply that oogenesis or oocyte maturation is stimulated by mating.
Analysis of the rate of egg laying by mated hermaphrodites showed that the increase in progeny production is due to an increase in the duration of production of progeny rather than an increase in the rate. It was also observed that old wild-type hermaphrodites that have ceased laying oocytes can be stimulated to continue oocyte production by mating. Several temperature-sensitive mutants have been isolated that lay very few oocytes at restrictive temperature. Many of these can be stimulated to produce large numbers of progeny if mated by males (Hirsh and Vanderslice, 1976; Nelson et al., 1978; D. Hirsh and L. Edgar, personal communication; J. Miwa and S. Ward, unpublished observations). At least one of these mutants
316
DEVELOPMENTAL
BIOLOGY
VOLUME
TABLE THE
EFFECT
OF MATING
N Unmated control Mated, >0.8 outcross Mated, no outcross
10 5 17
2
ON PROGENY
Progeny/worm 199 + 10 479 f 84 163 -t 24
73,1979
AND OOCYTE P
