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Diploidy is a fundamental genetic feature in mammals, in which haploid cells normally arise only as post-meiotic germ cells that serve to ensure a diploid genome upon fertilization. Gamete manipulation has yielded haploid embryonic stem (ES) cells from several mammalian species, but haploid human ES cells have yet to be reported. Here we generated and analysed a collection of human parthenogenetic ES cell lines originating from haploid oocytes, leading to the successful isolation and maintenance of human ES cell lines with a normal haploid karyotype. Haploid human ES cells exhibited typical pluripotent stem cell characteristics, such as self-renewal capacity and a pluripotency-specific molecular signature. Moreover, we demonstrated the utility of these cells as a platform for loss-of-function genetic screening. Although haploid human ES cells resembled their diploid counterparts, they also displayed distinct properties including differential regulation of X chromosome inactivation and of genes involved in oxidative phosphorylation, alongside reduction in absolute gene expression levels and cell size. Surprisingly, we found that a haploid human genome is compatible not only with the undifferentiated pluripotent state, but also with differentiated somatic fates representing all three embryonic germ layers both in vitro and in vivo, despite a persistent dosage imbalance between the autosomes and X chromosome. We expect that haploid human ES cells will provide novel means for studying human functional genomics and development.

Fertilization occurs when sperm and egg recognize each other and fuse to form a new, genetically distinct organism. The molecular basis of sperm-egg recognition is unknown, but is likely to require interactions between receptor proteins displayed on their surface. Izumo1 is an essential sperm cell-surface protein, but its receptor on the egg has not been described. Here we identify folate receptor 4 (Folr4) as the receptor for Izumo1 on the mouse egg, and propose to rename it Juno. We show that the Izumo1-Juno interaction is conserved within several mammalian species, including humans. Female mice lacking Juno are infertile and Juno-deficient eggs do not fuse with normal sperm. Rapid shedding of Juno from the oolemma after fertilization suggests a mechanism for the membrane block to polyspermy, ensuring eggs normally fuse with just a single sperm. Our discovery of an essential receptor pair at the nexus of conception provides opportunities for the rational development of new fertility treatments and contraceptives.

Most animals are diploid, but haploid-only and male-haploid (such as honeybee and ant) species have been described. The diploid genomes of complex organisms limit genetic approaches in biomedical model species such as mice. To overcome this problem, experimental induction of haploidy has been used in fish. Haploid development in zebrafish has been applied for genetic screening. Recently, haploid pluripotent cell lines from medaka fish (Oryzias latipes) have also been established. In contrast, haploidy seems less compatible with development in mammals. Although haploid cells have been observed in egg cylinder stage parthenogenetic mouse embryos, most cells in surviving embryos become diploid. Here we describe haploid mouse embryonic stem cells and show their application in forward genetic screening.

The existence of an all-female species of whiptail lizard, formed as a hybrid between sexual species, has been known since 1962. But how it produces mature eggs with a full somatic chromosomal complement has remained unclear, as has a mechanism for maintaining genetic diversity. Lutes et al. now show that in this species (Aspidoscelis tesselata) meiosis initiates with twice the number of chromosomes, compared to sexual species, and that pairing and recombination take place between genetically identical sister chromosomes instead of between homologues. The existence of all-female species of whiptail lizard, formed as a hybrid between sexual species, has been known since 1962; however, how the meiotic program is altered to produce diploid eggs while maintaining heterozygosity has remained unclear. Here it is shown in parthenogenetic species that meiosis initiates with twice the number of chromosomes compared to sexual species, and that pairing and recombination takes place between genetically identical sister chromosomes instead of between homologues. Although bisexual reproduction has proven to be highly successful, parthenogenetic all-female populations occur frequently in certain taxa, including the whiptail lizards of the genus Aspidoscelis. Allozyme analysis revealed a high degree of fixed heterozygosity in these parthenogenetic species1,2, supporting the view that they originated from hybridization events between related sexual species. It has remained unclear how the meiotic program is altered to produce diploid eggs while maintaining heterozygosity. Here we show that meiosis commences with twice the number of chromosomes in parthenogenetic versus sexual species, a mechanism that provides the basis for generating gametes with unreduced chromosome content without fundamental deviation from the classic meiotic program. Our observation of synaptonemal complexes and chiasmata demonstrate that a typical meiotic program occurs and that heterozygosity is not maintained by bypassing recombination. Instead, fluorescent in situ hybridization probes that distinguish between homologues reveal that bivalents form between sister chromosomes, the genetically identical products of the first of two premeiotic replication cycles. Sister chromosome pairing provides a mechanism for the maintenance of heterozygosity, which is critical for offsetting the reduced fitness associated with the lack of genetic diversity in parthenogenetic species.

Examination of the geographic distributions of sexual organisms and their asexual, or parthenogenetic, competitors reveals certain consistent patterns. These patterns are called geographic parthenogenes is1, 2, 3, 4, 5, 6, 7, 8. For example, if we compare sexual organisms with closely related asexuals, we find that, in the Northern Hemisphere, there is a strong tendency for the asexuals to occur further to the north. One researcher to document this pattern is Bierzychudek, who examined 43 cases (drawn from 10 genera) where the geographic distributions of a sexual plant and a closely related asexual are known4. In 76% of these cases, the asexual plant's range was more northerly than the range of the sexual. Some of the remaining cases probably fit with this pattern, but more data must be obtained before this suggestion can be confirmed. Asexuals also tend to occur at high altitudes, and in marginal, resource-poor environments1, 2, 3, 4, 5, 6, 7, 8. We have constructed a mathematical model of a habitat that stretches from south to north in the Northern Hemisphere. Our computer simulations based on this model support the idea that a single basic process may account for much of what is known about geographic parthenogenesis. This process involves the movement of individuals from areas in which they are well adapted to areas where they are poorly adapted.

THE large wingless Tettigoniid grasshopper Saga pedo (serrata) is an inhabitant of southern Europe, its distribution ranging from Spain to the Ural Mountains. Among the northern outposts of its range are some localities in the Moravian mountains and Voronij, Saratov and Ufa in Russia. It is remarkable that this species, which occurs farther north than any other representative of the genus, appears to reproduce normally by parthenogenesis. The biology and cytology of Saga pedo was studied by Matthey1 in material from the Swiss canton Valais. He found that the chromosome number of the parthenogenetic females generally amounted to 68, made up of six pairs of metacentric and twenty-eight pairs of acrocentric elements. This high number is unique among the Tettigoniidae, the idiograms of which range from 22 to 36 in all other species investigated. Matthey suggested, therefore, that S. pedo must in reality be a tetraploid.

THE cortical layer of the unfertilized egg of Psammechinus miliaris exhibits a remarkable positive birefringence (with respect to the radial axis) which has been interpreted as meaning that the cortical layer is built up of radially arranged lipid molecules1,2. The birefringence disappears at fertilization and reappears during the anaphase of the first mitosis3. This means that the cortical layer undergoes rhythmic variations of its submicroscopic structure. We have investigated the cycle of such structural variations in relation to the phases of mitosis and to the modifications of permeability occurring during the early stages of development, in both fertilized and parthenogenetic eggs.

Apomixis, the formation of asexual seeds in plants, leads to populations that are genetically uniform maternal clones. The transfer of apomixis to crop plants holds great promise in plant breeding for fixation of heterozygosity and hybrid vigour because it would allow the propagation of hybrids over successive generations. Apomixis involves the production of unreduced (diploid) female gametes that retain the genotype of the parent plant (apomeiosis), followed by parthenogenetic development of the egg cell into an embryo and the formation of functional endosperm. The molecular mechanisms underlying apomixis are unknown. Here we show that mutation of the Arabidopsis gene DYAD/SWITCH1 (SWI1), a regulator of meiotic chromosome organization, leads to apomeiosis. We found that most fertile ovules in dyad plants form seeds that are triploid and that arise from the fertilization of an unreduced female gamete by a haploid male gamete. The unreduced female gametes fully retain parental heterozygosity across the genome, which is characteristic of apomeiosis. Our results show that the alteration of a single gene in a sexual plant can bring about functional apomeiosis, a major component of apomixis.

Parthenogenesis, the production of offspring without fertilization by a male, is rare in vertebrate species, which usually reproduce after fusion of male and female gametes. Here we use genetic fingerprinting to identify parthenogenetic offspring produced by two female Komodo dragons (Varanus komodoensis) that had been kept at separate institutions and isolated from males; one of these females subsequently produced additional offspring sexually. This reproductive plasticity indicates that female Komodo dragons may switch between asexual and sexual reproduction, depending on the availability of a mate--a finding that has implications for the breeding of this threatened species in captivity. Most zoos keep only females, with males being moved between zoos for mating, but perhaps they should be kept together to avoid triggering parthenogenesis and thereby decreasing genetic diversity.

Even though the genetic fingerprint of human sperm has been defined, its role in orchestrating fertilization and the development of the early embryo remains vague. Here we show that human male gametes pass over more to the oocyte than just the haploid male genome--paternal messenger RNAs are also delivered to the egg at fertilization. If these transcripts, previously thought to be left-overs from spermatogenesis, are important in early development, our findings may have implications for the success of somatic-cell nuclear transfer in cloning technology and the identification of components leading to unexplained male-factor infertility.

Only mammals have relinquished parthenogenesis, a means of producing descendants solely from maternal germ cells. Mouse parthenogenetic embryos die by day 10 of gestation. Bi-parental reproduction is necessary because of parent-specific epigenetic modification of the genome during gametogenesis. This leads to unequal expression of imprinted genes from the maternal and paternal alleles. However, there is no direct evidence that genomic imprinting is the only barrier to parthenogenetic development. Here we show the development of a viable parthenogenetic mouse individual from a reconstructed oocyte containing two haploid sets of maternal genome, derived from non-growing and fully grown oocytes. This development was made possible by the appropriate expression of the Igf2 and H19 genes with other imprinted genes, using mutant mice with a 13-kilobase deletion in the H19 gene as non-growing oocytes donors. This full-term development is associated with a marked reduction in aberrantly expressed genes. The parthenote developed to adulthood with the ability to reproduce offspring. These results suggest that paternal imprinting prevents parthenogenesis, ensuring that the paternal contribution is obligatory for the descendant.

Wolbachia (Rickettsiaceae) is a genus of maternally inherited endosymbiotic bac-teria commonly found in the reproductive tissues of arthropods. These bacteria manipulate host reproduction to increase the number of infected individuals within the population, erecting intraspecific fertility barriers, causing parthenogenesis or resulting in the feminization of genetic males1. They are usually transmitted vertically, so we predicted that they should have evolved a mechanism to target the host's germ cells during development. Here we show that Wolbachia become concentrated in the germ plasm of the Drosophila egg. Mutations in developmental patterning genes2 demonstrate that this localization is dependent on the assembly of germ plasm.

The discovery of an all-female population of damselflies in the Azores archipelago provides a novelty for entomologists. It also highlights the unique selection pressures faced by species that colonize islands.

THE c-mos proto-oncogene encodes a 37-39K cytoplasmic serine/ threonine kinase1 implicated in the meiotic maturation events during murine spermatogenesis2 and oogenesis3–6. In Xenopus, ectopic expression of pp39mos can promote both the meiotic matur-ation of oocytes7–9 and also arrest the cleavage of blastomeres10. To elucidate the role of pp39mos we have generated homozygous mutant mice by gene targeting in embryonic stem cells11. These mice are viable and mutant males are fertile, demonstrating that pp39mos is not essential for spermatogenesis. In contrast, mutant females, have a reduced fertility because of the failure of mature eggs to arrest during meiosis. c-mos-/- oocytes undergo germinal vesicle breakdown and extrusion of both polar bodies followed in some cases by progression into cleavage. Mutant females also develop ovarian cysts. These results demonstrate that a major role for pp39mos is to prevent the spontaneous parthenogenetic activation of unfertilized eggs.

Although we do not know why sex evolved, the twofold cost of meiosis for females provides a standard against which postulated benefits of sex can be evaluated. The most reliable benefit is sex's ability to reduce the impact of deleterious mutations. But deleterious mutations may themselves generate a large and previously overlooked female-specific cost of sex. DNA sequence comparisons have confirmed Haldane's suggestion that most mutations arise in the male germ line; recent estimates of a, the ratio of male to female mutation rates, are ten, six and two in humans, primates and rodents, respectively. Consequently, male gametes may give progeny more mutations than the associated sexual recombination eliminates. Here I describe computer stimulations showing that the cost of male mutations can easily exceed the benefits of recombination, causing females to produce fitter progeny by parthenogenesis than by mating. The persistence of sexual reproduction by females thus becomes even more problematic.

Two classes of models attempt to explain why obligate parthenogenesis only rarely replaces sexual reproduction in natural populations, in spite of the apparent reproductive advantage that parthenogens gain by producing only female offspring1. The mutation-accumulation models suggest that sex is adaptive because it purges the genome of harmful recurrent mutations2,3. The ecological genetic models postulate that sex is adaptive in variable environments, particularly when the relevant variation is generated by revolutionary interactions with parasites4–7. Both of these models have considerable merit, but would seem to have limitations. The mutation-accumulation models require high rates of mutation3,8; the coevolutionary models require that parasites have severe fitness effects on their hosts9. In addition, parasites could select for clonal diversity and thereby erode any advantage that sex gains by producing variable progeny10. Here we consider the interaction between mutation accumulation and host–parasite coevolution. The results suggest that even moderate effects by parasites combined with reasonable rates of mutation could render sex evolutionary stable against repeated invasion by clones.

Inherited rickettsial symbionts of the genus Wolbachia occur commonly in arthropods and have been implicated in the expression of parthenogenesis, feminization and cytoplasmic incompatibility Wolbachia from the Asian tiger mosquito, Aedes albopictus, to replace the natural infection of Drosophila simulans by means of embryonic microinjection techniques. The transferred Wolbachia infection behaves like a natural Drosophila infection with regard to its inheritance, cytoskeleton interactions and ability to induce incompatibility when crossed with uninfected flies. The transinfected flies are bidirectionally incompatible with all other naturally infected strains of Drosophila simulans, however, and as such represent a unique crossing type. The successful transfer of this symbiont between distantly related hosts suggests that it may be possible to introduce this agent experimentally into arthropod species of medical and agricultural importance in order to manipulate natural populations genetically.

Wolbachia, intracellular bacteria transmitted through the egg, have been estimated to infect more than 16% of all insect species, as well as other arthropods1. They distort their hosts' reproduction, inducing parthenogenesis, feminization and cytoplasmic incompatibility2. This favours the reproduction of infected female hosts at the expense of uninfected females. Here we show that several Wolbachia strains that cannot generate modifications in host sperm can still rescue the modifications caused by other strains as long as the two strains are sufficiently closely related.

It has been rumoured1 that an unidentified decapod crustacean, a crayfish of marbled appearance and of uncertain geographical origin that was introduced into the German aquarium trade in the mid-1990s, is capable of unisexual reproduction (parthenogenesis). Here we confirm that this marbled crayfish ('Marmorkrebs') is parthenogenetic under laboratory conditions and use morphological and molecular analysis to show that it belongs to the American Cambaridae family. Although parthenogenesis is widespread among the Crustacea2, and shrimp, lobsters, crayfish and crabs are otherwise versatile in their modes of reproduction3,4,5, it has not been reported before in decapods, the largest and economically most important crustacean group. By virtue of its parthenogenetic reproduction, the marbled crayfish emerges not only as an interesting laboratory model but also as a potential ecological threat in that it could outcompete native forms should even a single specimen be released into European lakes and rivers.

A CENTRAL paradigm in evolutionary biology is that sexual reproduction is advantageous over asexuality1–5. One of the long-term disadvantages asexual forms have to face is Muller's ratchet6. In the absence of recombination, theoretically no genotype can ever produce offspring with fewer mutations than its own load. The accumulation of deleterious mutations and gene combinations that cannot be purged should lead to extinction of parthenogenetic forms within 104–105 generations7,8. Evidence is accumulating, however, that some of these might have survived for such periods or even longer9–14. In the Amazon Molly fish Poecilia Formosa we have detected a process that appears to compensate for disadvantages of asexuality, namely incorporation of subgenomic amounts of DNA from a bisexual host species by microchromosomes.

Nature 370, 68-71 (1994) FIGURE 3b and c of this Letter was an early version that should not have been published. The correct version of this figure is shown here.

EIGHT years ago I discovered in the Quebrada (i.e. ravine) of Guarenas, about nine miles to the east of Caracas, and approximately at 650 metres above the level of the sea, one of those charming groups of tropical vegetation, which are equally interesting to the botanist and to the lover of the picturesque beauties of nature. A magnificent specimen of Pogonopus Ottonis was all aglow with its large rosy sepals; up to its highest branches a luxuriant Vitis caribœa had ascended in graceful festoons, laden with blackish grapes, and displaying now and then in the gentle breeze the silvery glimmer from the under side of its palmate leaves; whilst in the damp shade underneath throve a colony of Gloxinia pallidiflora, a plant which from its bruised leaves gives out a smell identical with that of the spearmint.

The presence of both parental genomes is essential for development to term in the mouse embryo1–5 probably because of germline-specific modifications of homologous chromosomes6,7. Neither androgenetic nor parthenogenetic embryos can by themselves develop to term; any post-implantation embryos they produce have opposite phenotypes, which reflects the presence of complementary information in parental chromosomes1,7,8. The development of androgenetic ↔ parthenogenetic chimaeras is of considerable interest because in this case both parental chromosomes are available even though they are in separate cells. We demonstrate here that in post-implantation chimaeric fetuses, the expression of parental information results in spatial specificity so that parthenogenetic cells are confined to the embryo but the trophoblast consists almost entirely of androgenetic cells. The yolk sac contains both cell types. However, there is incomplete functional complementation because the chimaeras do not reach term. Although failure to reach term may occur partly because of inadequate intermingling and interactions between embryonic cells, it is more likely that further control of mouse development depends on the presence of both sets of chromosomes within the same cells.

OOCYTES of many species undergo parthenogenetic activation after exposure to the calcium ionophore, A23187, even in the absence of external Ca2+ (refs 1 and 2), whereas those of mouse, rat and hamster will activate simply on exposure to Ca2+ free medium3,4. This suggests that Ca2+ is involved in parthenogenetic activation. Direct evidence of a transient increase in intracellular free Ca2+ at the time of fertilisation or activation of fish5 and sea urchin eggs6 has been obtained using the photoprotein aequorin. We report here that parthenogenetic activation and subsequent normal development can be induced in mouse oocytes by injecting them with Ca2+.

WE have previously exposed sea urchin eggs to micromolar amounts of the divalent transporting ionophore A23187, and observed every parameter of normal fertilisation. The cortical reaction with elevation of the fertilisation membrane, the plasma membrane conductance changes, the respiratory burst, and increases in protein and DNA synthesis were all initiated in the usual fashion1. These activations of Lytechinus pictus and Strongylocentrotus purpuratus eggs were independent of the ionic composition of the external solutions1. A23187 seemed to act by releasing intracellular Ca2+, for eggs preloaded with 45Ca showed a twentyfold increase in 45Ca-efflux when activated by ionophore or fertilisation1. Measurements of free and bound calcium and magnesium in homogenates of unfertilised eggs showed that most of the Mg2+ was already available in soluble form, whereas the Ca2+ was sequestered but available for release1. After we found that 50 µM A23187 could activate eggs of the mollusc Acmaea insessa and the protochordate Ciona intestinalis to undergo several abnormal cleavages, we wondered whether ionophore activation might be a general phenomenon. To our knowledge artificial parthenogenesis has not been obtained with Acmaea and Ciona eggs by other methods. We have now found that calcium ionophore activation is common to several species widely separated phylogenetically.

The widespread occurrence of sexual reproduction is an important problem in evolutionary theory, because of the cost of producing males in dioecious organisms and the allocation of limited resources in cosexes1–4. Here I compare the predictions of the major ecological hypotheses on the maintenance of sex by examining male frequency in Potamopyrgus antipodarum, a dioecious freshwater snail with both sexual and parthenogenetic populations5. The results do not-support the view that sex is maintained by a variable physical environment6–9, but they are consistent with the idea that sex is favoured by selection resulting from host–parasite interactions10–14.

Parthenogenesis occurs spontaneously in about 10% of ovulated eggs of inbred strain LT/Sv mice (Stevens and Varnum 1974; Stevens 1975) and is experimentally inducible in other strains by various physical and chemical agents (see Tarkowski 1975 for review). The development of most parthenotes seems normal up to the expanded blastocyst stage (Van Blerkom and Runner 1976). They implant in the uterus, but, for reasons unknown, are resorbed within a few days. Although they rarely survive to 8 days of gestation when they develop somites, heart muscle, amnion, and neuroepithelium, Kaufman, Barton, and Suram (1977) obtained two embryos with 25 somites by transferring parthenogenetic blastocysts to the uteri of ovariectomised females treated with exogenous hormones. Even though parthenogenetic embryos do not survive to birth, their cells contain genetic information that permits prolonged survival. If two-cell parthenotes are cultured to the blastocyst stage and then grafted to extrauterine sites such as the testis or kidney, they may survive as teratomas composed of several types of tissues and undifferentiated embryonal cells (Stevens 1974; Ilse et al. 1975; and L.C.S. and D.S.V., unpublished).

It has been suggested that the failure of parthenogenetic mouse embryos to develop to term is primarily due to their aberrant cytoplasm and homozygosity leading to the expression of recessive lethal genes. The reported birth of homozygous gynogenetic (male pronucleus removed from egg after fertilization) mice and of animals following transplantation of nuclei from parthenogenetic embryos to enucleated fertilized eggs, is indicative of abnormal cytoplasm and not an abnormal genotype of the activated eggs. However, we and others have been unable to obtain such homozygous mice. We investigated this problem further by using reconstituted heterozygous eggs, with haploid parthenogenetic eggs as recipients for a male or female pronucleus. We report here that the eggs which receive a male pronucleus develop to term but those with two female pronuclei develop only poorly after implantation. Therefore, the cytoplasm of activated eggs is fully competent to support development to term but not if the genome is entirely of maternal origin. We propose that specific imprinting of the genome occurs during gametogenesis so that the presence of both a male and a female pronucleus is essential in an egg for full-term development. The paternal imprinting of the genome appears necessary for the normal development of the extraembryonic membranes and the trophoblast.

SEA anemones of the genus Actinia seem to be dioecious and reproduce sexually, yet males, females, and individuals without gonads brood juveniles for much of the year1–3. How this occurs is still not understood and knowledge on the subject is scant. Gillespie4 considered that eggs develop to ciliated blastulae and then directly into “a cylindrical form … which develops tentacular buds”. Chia and Rostron3 found no pre-planula larval stages in the coelen-terons of hundreds of specimens they examined and postulated that most embryos must leave the mother as morulae or blastulae and later resettle in the coelenteric cavities of other adult A. equina where they metamorphose to post-Edwardsia juveniles. According to this hypothesis, juveniles contained in an adult should be derived from a number of different parents and would be expected to show a range of phenotypes characteristic of the population rather than the brooding adult. Cain5 however found that in phenotypically varied populations of A. equina young anemones invariably had the same colour as the adult from which they were released. He considered it likely that A. equina is a protandric hermaphrodite which mainly self-fertilises and retains larvae within the parent, even though Carlgren2 thought this “hardly probable”.