- Research article
- Open Access
Oocyte production and sperm utilization patterns in semi-fertile strains of Caenorhabditis elegans
© Kadandale and Singson; licensee BioMed Central Ltd. 2004
Received: 04 March 2004
Accepted: 15 April 2004
Published: 15 April 2004
Caenorhabditis elegans hermaphrodites are capable of producing hundreds of progeny. However, genetic and environmental factors can keep many animals from attaining their full reproductive potential. In these situations, efficient use of any functional gametes becomes more important for reproductive success. To learn about this aspect of C. elegans reproductive biology, we examined oocyte production and sperm utilization patterns in a unique collection of semi-fertile sperm function mutants.
In the mutants examined here, broods can be very small but sperm induced high levels of ovulation. Ovulation rates reach maximum levels between the first and second day of adulthood. Ovulations rates remain high during the reproductive period and gradually decline with age. These results further demonstrate a decoupling of the ability of sperm to fertilize oocytes and induce ovulation. We also observe that in our semi-fertile mutants the peak of successful fertilization events precedes the bulk of oocyte production. Mixing populations of functional and nonfunctional sperm under conditions without sperm competition also shows that functional sperm are utilized efficiently. Although overall brood size can be similar for different mutant strains, slight differences in the pattern of sperm utilization in these strains can lead to significant differences in resource utilization and population growth.
This study represents the first detailed description of oocyte and progeny production patterns over the entire reproductive period for wild-type and fertility impaired strains of C. elegans. The phenotype of our mutants provide an ideal system for studying sperm utilization patterns since they only affect one major process, the ability to fertilize oocytes. In semi-fertile mutants, the nature of the reproductive process and/or specific molecular mechanisms ensures that any functional sperm are utilized quickly. Only a fraction of the sperm produced by our semi-sterile mutants are functional as opposed to every sperm having a low but equal chance of fertilizing an oocyte. In addition to the number of progeny produced, the pattern of progeny production can have an important influence on the dynamics of population growth.
The C. elegans strain considered "wild type" – N2, exists primarily in the hermaphroditic condition, although males are produced spontaneously (due to chromosomal nondisjunction) at an extremely low frequency [1, 2]. During its fourth (and final) larval stage, the reproductive system of the hermaphrodite produces its lifetime's supply of sperm . As development progresses, the germline undergoes a switch, and for the remainder of the animal's reproductive life will produce only oocytes . In this organism, it is not the supply of oocytes which limits the number of progeny that each hermaphrodite produces, but rather, the number of functional sperm that are made in the larval stage [4, 5].
When hermaphrodites are mated to males, male sperm will outcompete the hermaphrodite's own sperm and will preferentially fertilize the oocytes . This phenomenon is referred to as sperm competition, and in the nematode C. elegans specifically refers to the competition effects seen between male and hermaphrodite sperm. Unlike in other species, C. elegans exhibits no competition between sperm from different males . Hermaphrodites mated to multiple males show no male precedence effects. The ability of male-derived sperm to outcompete hermaphrodite-derived sperm stems from intrinsic physiological differences between male and hermaphrodite sperm. It has been shown that male sperm are larger than hermaphrodite sperm, and that this size difference is important in the context of sperm competition in C. elegans [9–11]. Further, the ability of male sperm to outcompete hermaphrodite sperm has been shown to be independent of their ability to fertilize the oocyte .
A class of mutations has been defined that result in the production of sperm that are incapable of fertilizing the oocyte [5, 13]. This is despite that fact that mutant sperm are produced in normal quantities, appear morphologically normal, activate to form crawling spermatozoa, localize correctly to the spermatheca and come in contact with oocytes [5, 14]. Thus, these mutations seem to affect molecules that are required by the sperm specifically for fertilization and not for sperm development. These mutants are collectively called the spe-9 class, after the first of the corresponding genes to be cloned. While some alleles of these genes are completely sterile at all temperatures, others show temperature-sensitive sterility and still others show partial sterility (leaky sterile) at various culture conditions (Kadandale & Singson, unpublished observations).
Results and discussion
Wild-type sperm utilization and ovulation patterns
Sperm utilization patterns in leaky spe-9 class mutants
Brood sizes of various mutants used in this study.
spe-9(eb23); dpy-1, smg-6
Two-male mate experiments
Food clearing assay
Developmental time is the same for two spe-9-class mutants used in resource utilization assay.
64.5 ± 0.6
64.6 ± 0.5
In order to rapidly utilize functional sperm, it is reasonable to assume that the oocyte must have some means of distinguishing these, since it comes in contact, concurrently, with almost the whole population of sperm within the spermatheca (Fig. 1). The molecular components encoded by genes like spe-40 could influence the efficiency of this process. While our assays only measure quantitative differences in resource utilization, we can also imagine how such a method of discriminating between populations of sperm can also be used to produce qualitatively different offspring.
There appears to be a robust mechanism that operates within the reproductive tract of the C. elegans hermaphrodite that ensures that functional sperm are utilized quickly. Efficient sperm storage and presentation to the newly ovulated oocyte is likely to contribute to this efficiency and is an inherent feature of worm reproductive biology. In our mutants, only a fraction of the sperm produced by each worm are functional. Further, any functional sperm are used efficiently in all strains tested. However, small variations in this efficiency exits between strains. These small variations in sperm usage patterns can lead to significant differences in population growth, dynamics and resource utilization. The underlying molecular events and mechanism involved could have important influences on sexual selection and the reproductive success of animals in the wild.
fer-14(hc14), spe-13(hc137), and spe-40(it133) are all spe-9 class mutants that have not yet been cloned . fem-1(hc17) has been described elsewhere . Several alleles of spe-9 are not completely sterile . The spe-9(eb23); dpy-1(e1) strain has a larger brood size (Kadandale & Singson, unpublished observations) when in the background of a smg-6 mutation. smg-6 is known to be required for mRNA surveillance and nonsense mediated decay [19–21]. The observation that the fertility of the eb23 allele increases in the smg background suggests that the semi-fertility of these mutants is due to read-through translation. spe-9(eb19); ebEx149 is a strain of worms that carry a transgene bearing the cosmid C17D12 and the dominant phenotypic marker, rol-6. Although C17D12 rescues spe-9 mutants, it has a truncated form of spe-9 that lacks the cytoplasmic tail and synthesizes less SPE-9 than can be detected by antibody staining (Zannoni and Singson, unpublished observations). Germline expression of transgenic extrachromosomal arrays is known to be poor [22, 23], and this could explain why so little SPE-9 is made in these worms. The spe-9(eb19); him-5; ebEx149 strain carried the him-5 mutation. This mutation causes males to be produced at high frequency, but does not have any adverse effects on sperm . At least 10 animals were used for all brood, ovulation and developmental time experiments (see below).
Sperm utilization patterns
Worms homozygous for the required mutation were grown up as a stock according to standard protocols and culture conditions (20°C see below) . 20–30 young adult worms were picked to fresh plates and allowed to lay eggs for one hour. In cases where the homozygous stock did not produce sufficient progeny in one hour, heterozygotes were picked, and only their sterile progeny (which would be homozygous for the desired mutation) were used. The adult worms were then removed and the eggs were allowed to hatch and develop until they reached the L4 larval stage. At this time the worms were picked individually to fresh plates. The worms were transferred to fresh plates every twelve hours and the number of fertilized eggs and unfertilized oocytes that had been laid were counted for each worm. The first twelve-hour period in which either fertilized or unfertilized eggs were seen was taken as the 0–12 hour time point. Since L4 larvae were picked and had to still initiate zygote production, there was typically a short lag in ovulation rates for the 0–12 hour time point. The mutants used in this study were chosen since their semi-fertility was not completely temperature-sensitive, so that all brood sizes, sperm utilization patterns and total ovulation patterns could be checked at 20°C and compared relative to each other. Total ovulations were calculated as the sum of fertilized eggs and unfertilized oocytes laid at each time point.
Sequential two male mates
These were performed essentially as described . In short, single fem-1(hc17) animals were picked to fresh plates and each was mated to four spe-9(eb19); him-5 males for three hours. The males were then removed and the fem-1 animals were allowed to lay eggs for one hour, after which they were transferred to fresh plates. Successful mating was scored by looking for an increase in the basal ovulation rate of fem-1 hermaphrodites. Half of these were not second-mated to any males, to check for persistence of sperm for the duration of the assay. The remaining animals were mated to four N2 males each for three hours. The males were removed and the hermaphrodites were allowed to lay eggs for one hour. The plates were checked for fertilized eggs or unfertilized oocytes.
Plate clearing assay
The plate clearing assay was modified from the method previously described . Every plate was seeded with an equal amount of bacterial (E. coli – OP50) culture. Either two worms (spe-40(it133), fer-14(hc14) or N2) or one worm (fer-14) of each strain was picked to these plates as L4 animals and the time taken for each plate to be cleared of food was noted.
Heterozygous fer-14(hc14)/+ or spe-40(it133)/+ animals were picked and allowed to lay eggs for one hour. The adult worms were then picked off and the eggs allowed to hatch. Once they hatched, the young worms were transferred to individual plates and the time taken for them to lay their first egg/unfertilized oocyte was noted. Animals were scored as homozygous mutants or wild type (or heterozygotes) based on their fertility.
We would like to thank Chi-hua Chiu, Jody Hey, Monica Driscoll, Mohamed Noor and Iris Dihlmann for critical comments on the manuscript. We thank members of the lab for helpful discussions. The Caenorhabditis Genetics Center provided some nematode strains, and it is funded by the National Institutes of Health National Center for Research Resources (NCRR). Work in the lab was supported by a grants to A.W.S. from the National Institutes of Health (R01 GM63089-01), the National Science Foundation (IBN-0000182), and a Charles & Johanna Busch Biomedical Fund. P.K. was supported by a Charles & Johanna Busch fellowship in molecular biology.
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