Preventing aneuploidy: The groom must wait until the bride is ready

Fertilization often triggers the final step of haploidization of the female gamete genome. In this issue, Mori et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202012001) identify two successive actin-dependent mechanisms that delay fusion of maternal and paternal chromosomes, preventing inadvertent elimination of paternal chromosomes together with maternal ones.

Species propagating via sexual reproduction produce haploid gametes through meiosis, a mode of division that culminates with two consecutive rounds of divisions with no intervening DNA replication. Fusion of the two haploid gametes, the sperm and the egg, restores the ploidy of the species. In most mammals, fertilization engages the second round of sister chromatid division of the egg, the unwanted chromosomes being extruded into a small degenerating polar body. In this issue, Mori et al. (1) identify mechanisms that prevent the inadvertent encounter of male and female chromosomes until the mouse egg finishes its second meiotic division. These processes are essential to avoid inadvertent elimination of the male genome together with the haploid maternal set.Mouse eggs are ovulated while arrested in metaphase of the second meiotic division (metaphase II). Fertilization triggers meiosis resumption and anaphase II. It has been observed that the binding of the sperm is favored where the metaphase II–arrested egg is covered with microvilli (2) and disfavored in the region above the second meiotic spindle, named the actin cap, which is enriched in F-actin and devoid of microvilli (Fig. 1 A; 3). However, the mechanisms behind these observations have been poorly investigated. By following the early events of sperm binding to the egg, Mori et al. discover potential new roles for two membrane proteins previously implicated in sperm/egg binding, namely Juno (4) and CD9 (5, 6, 7).Open in a separate windowFigure 1.Sperm binding is favored outside the maternal actin cap for successful embryo development. The density of microvilli (bold red area) increases away from the metaphase II egg spindle, in correlation with more important Juno and CD9 staining patterns. (A) Sperm binding outside the maternal actin cap. A sperm bound in the transition zone (lighter red area), with fewer microvilli and more lamellipodia-like structures, will move toward the denser microvilli area where it will fuse to the egg, preventing its elimination during second polar body extrusion. This mechanism favors correct ploidy of the zygote and its development to term. (B) Sperm binding inside the maternal actin cap. Binding and fusion in the microvilli-depleted region will produce aneuploid zygotes with reduced chances of successful development. Maternal chromosomes appear in pink, paternal ones in blue, parental genome mixing in purple, and spindle microtubules in green.In a precise follow-up of sperm entry sites, the authors confirm previous observations that the fusion of the sperm occurs in a region >24 µm away from the maternal chromosomes. Simulations acknowledge a clear deviation of the in vivo sperm entry sites from a random configuration. Live imaging uncovers a rapid drift in the sperm’s peripheral position after its entry into the egg. These observations argue that not only does the sperm bind apart from the maternal chromosomes, but that it is also rapidly moved away to prevent further mixing while the second meiotic spindle rotates and the egg becomes truly haploid. This raises two questions that the authors address here: (a) how is the favored binding site—far away from the maternal spindle—determined, and (b) how is the sperm shifted from its original position once fused to the egg?Mori et al. (1) first show that two key proteins in sperm fusion to the egg, Juno (4) and CD9 (5, 6, 7), display a punctate distribution on the egg membrane with a density correlating with that of the microvilli; greater away from maternal chromosomes and lower above the maternal set. Intriguingly, Juno and CD9 are also abundant in the transition zone, rich in lamellipodia-like structures, between the microvilli-poor and -dense regions. In this transition zone, Juno and CD9’s labeling is dynamic, moving toward the denser microvilli area. When a fluorescently labeled sperm binds in this ring-like zone, it was observed to move away from maternal chromosomes before fusion to the egg. Conversely, if it binds in the dense microvilli portion of the egg, the sperm remains still (Fig. 1 A). Considering the major roles of Juno and CD9 in regulating sperm hooking, it is tempting to speculate that these two proteins promote sperm motion in the ring-like zone before its fusion, trafficking it away from the maternal genome.Next, the authors investigate the mechanisms behind Juno and CD9 local accumulation on the egg membrane and its potential role in regulating sperm binding. The region overhanging the maternal chromosomes is deprived of microvilli but enriched in F-actin (3). Owing in part to the fact that chromosomes accumulate the Ran GTPase in its GTP-bound active form, a Ran GTP gradient culminating at chromosomes is created around them (8). This Ran GTP gradient plays a major role in local F-actin accumulation and egg polarization (9). Not surprisingly, using an F-actin destabilizing drug as well as overexpression of a dominant negative form of the Ran GTPase, the authors observe that both F-actin and Ran control the accumulation of Juno and CD9 outside the F-actin cap overhanging maternal chromosomes. When the Ran GTP gradient is abolished, this bias in sperm fusion sites on the egg is no longer observed. If F-actin is disrupted using Latrunculin B, fusion sites occur closer to the chromosomes than in controls but do not appear to be random, arguing that F-actin may have other functions that require deeper investigation. The use of other tools to disrupt F-actin (e.g., cytochalasin D or Arp2/3 complex inhibitors) could help identify those F-actin–specific roles that are independent of Ran GTPase.Finally, the authors use intracytoplasmic sperm injection (ICSI) to introduce and position chromosomes at different locations inside the egg, allowing them to test the importance of this proposed sequestration away from maternal chromosomes until egg anaphase II is achieved. In about two thirds of the cases, having paternal chromosomes in a 20-µm proximity to maternal ones is deleterious for the one-cell zygote, since they are either eliminated into the second polar body or they fuse with the egg haploid set into a single pronucleus, preventing correct independent paternal genome preparation (Fig. 1 B). However, this is not observed when ICSI is performed 20 µm away from the maternal genome, thus demonstrating clearly the importance of sperm binding outside the maternal F-actin cortical cap. These ICSI experiments also show that in 20% of the cases, even if paternal chromosomes fuse inside the cortical cap area, they can be moved away by F-actin–dependent mechanisms.Altogether, Mori and colleagues (1) illustrate here the importance of delaying the fusion of mouse maternal and paternal genomes to ensure proper zygotic development. While the two pronuclei meet in center of the zygote via F-actin and microtubule-dependent mechanisms (10), it is only in interphase of the two-cell embryo that both parental genomes will potentially mix (11), attesting to the idea that this occurs quite late in the process. The authors show here that two mechanisms, sperm binding and sperm secondary motion inside the egg—both dependent on F-actin—promote the delay in genome fusion. Interestingly, the importance of F-actin in delaying the parental chromosome encounter is also observed in the Caenorhabditis elegans zygote (12), arguing that the process is evolutionary conserved. It will be interesting to determine whether Juno and CD9 also actively participate in egg polarization in a positive feedback loop or if they are only following egg polarity. This might be tested by forcing their localization at various points across the egg membrane, even in the cortical cap area. Ultimately, it might prove important to identify which regulators control their dynamic distribution in the ring-like transition zone on the egg membrane. It might be worth testing whether hydrodynamic forces triggered by the rotation of the second meiotic spindle participate in their oriented mobility toward the microvilli-dense region and maybe potentially in the sperm secondary motility (13), which remains to be further investigated.     

Kinesin-1 prevents capture of the oocyte meiotic spindle by the sperm aster

Centrioles are lost during oogenesis and inherited from the sperm at fertilization. In the zygote, the centrioles recruit pericentriolar proteins from the egg to form a mature centrosome that nucleates a sperm aster. The sperm aster then captures the female pronucleus to join the maternal and paternal genomes. Because fertilization occurs before completion of female meiosis, some mechanism must prevent capture of the meiotic spindle by the sperm aster. Here we show that in wild-type Caenorhabditis elegans zygotes, maternal pericentriolar proteins are not recruited to the sperm centrioles until after completion of meiosis. Depletion of kinesin-1 heavy chain or its binding partner resulted in premature centrosome maturation during meiosis and growth of a sperm aster that could capture the oocyte meiotic spindle. Kinesin prevents recruitment of pericentriolar proteins by coating the sperm DNA and centrioles and thus prevents triploidy by a nonmotor mechanism.     

Centrosome inheritance in starfish zygotes: selective loss of the maternal centrosome after fertilization

The mature egg inherits a centrosome from the second meiotic spindle, and the sperm introduces a second centrosome at fertilization. Since only one of these centrosomes survives to be used in development, specific mechanisms must exist to control centrosome inheritance. To investigate how centrosome inheritance is controlled we used starfish eggs as a model system, because they undergo meiosis after fertilization. As a result, the fate of the maternal and paternal centrosomes can be followed by light microscopy and experimentally manipulated in vivo. We show initially that only the paternal centrosome is used in starfish zygote development; the maternal centrosome retained from meiosis II is functionally lost before first mitosis. We then tested a number of possible ways in which the zygote could exert this differential control over the stability of centrosomes initially residing in the same cytoplasm. The results of these experiments can be summarized as follows: (1) Although the microtubule organizing center activity of the maternal centrosome is not degraded after meiosis, the ability of this centrosome to double at successive mitoses is lost. (2) The sperm centrosome is not "masked" from cytoplasmic conditions which could destabilize all centrosomes during or after the meiotic sequence. (3) The functional loss of the maternal centrosome is not due to its cortical location. (4) The loss of this doubling capacity is determined by the egg, not by putative inhibitory factors from the fertilizing sperm. (5) The destabilization of the maternal centrosome is not due to the complete loss of its centrioles. Together, these results demonstrate that all maternal centrosomes are equivalent and that they are intrinsically different from the paternal centrosome. This intrinsic difference, in concert with a change in cytoplasmic conditions after meiosis, determines the selective loss of the maternal centrosome inherited from the meiosis II spindle.     

The intimate genetics of Drosophila fertilization

The union of haploid gametes at fertilization initiates the formation of the diploid zygote in sexually reproducing animals. This founding event of embryogenesis includes several fascinating cellular and nuclear processes, such as sperm–egg cellular interactions, sperm chromatin remodelling, centrosome formation or pronuclear migration. In comparison with other aspects of development, the exploration of animal fertilization at the functional level has remained so far relatively limited, even in classical model organisms. Here, we have reviewed our current knowledge of fertilization in Drosophila melanogaster, with a special emphasis on the genes involved in the complex transformation of the fertilizing sperm nucleus into a replicated set of paternal chromosomes.     

Western white pine (Pinus monticola Dougl.) reproduction: II. Fertilisation and cytoplasmic inheritance

Fertilisation and proembryo development are described from transmission electron micrographs emphasising the origin and fate of the maternal and paternal mitochondria and plastids. During central cell and egg development mitochondria migrate toward the nuclei, forming a perinuclear zone consisting predominantly of maternal mitochondria and polysomes. At the same time, maternal plastids transformed and at fertilisation are excluded from the neocytoplasm. The pollen tube releases two sperm nuclei into the egg with cytoplasm from the generative cell and the tube cell. The leading sperm nucleus fuses with the egg nucleus and a small number of paternal mitochondria and plastids are taken into the perinuclear zone. The second sperm nucleus degenerates. As the zygote nucleus undergoes mitosis followed by free nuclear division and nuclear migration to the chalazal end of the archegonium, maternal and paternal organelles intermingle within the neocytoplasm. The result is paternal inheritance of plastids and biparental, but predominantly maternal, inheritance of mitochondria. This pattern is consistent within the Pinaceae but differs from some other conifer families. Received: 9 December 1999 / Revision accepted: 30 April 2000     

Cytological evidence of maternal meiotic errors in a line of chickens with a high incidence of triploidy.

Direct evidence of the nature of maternal meiotic errors in a selected line of chickens with a high incidence of triploidy was obtained by using cytologically marked paternal gametes derived from a closely related avian species. Matings were made by artificial insemination of female chickens of the selection line and a control line with semen from ring-necked male pheasants. A total of five triploid, one pentaploid, and 21 diploid hybrid embryos were karyotyped. Each triploid hybrid embryo contained one set of paternal pheasant chromosomes and two sets of maternal chicken chromosomes, providing irrefutable cytological evidence that the triploids were derived from diploid ova produced by females of the selection line. The pentaploid hybrid contained one set of paternal pheasant chromosomes and four sets of maternal chicken chromosomes, indicating that it had been derived from a tetraploid ovum. Females of the selection line are thought to have a genetically mediated susceptibility to nondisjunction which is responsible for the high incidence of meiotic errors. Evidence is provided that the non-disjunction occurs at both meiosis I and meiosis II.     

Mechanisms that prevent catastrophic interactions between paternal chromosomes and the oocyte meiotic spindle

Meiosis produces haploid gametes by accurately reducing chromosome ploidy through one round of DNA replication and two subsequent rounds of chromosome segregation and cell division. The cell divisions of female meiosis are highly asymmetric and give rise to a large egg and two very small polar bodies that do not contribute to development. These asymmetric divisions are driven by meiotic spindles that are small relative to the size of the egg and have one pole juxtaposed against the cell cortex to promote polar body extrusion. An additional unique feature of female meiosis is that fertilization occurs before extrusion of the second polar body in nearly all animal species. Thus sperm-derived chromosomes are present in the egg during female meiosis. Here, we explore the idea that the asymmetry of female meiosis spatially separates the sperm from the meiotic spindle to prevent detrimental interactions between the spindle and the paternal chromosomes.     

Early developmental processes in the fertilised honeybee (Apis mellifera) oocyte

The behaviour of sperm from egg penetration until creation of the zygote, the development of the maternal pronucleus, and the two first cleavage divisions were studied by use of fluorescence microscopy. It was found that 4-12 sperm penetrate the egg membranes prior to oviposition. Contrary to previous reports, we found that only 1-7 sperm move from their initial location just beneath the vitelline membrane and into the cytoplasm, where they develop into paternal pronuclei. At the time of oviposition, the oocyte nucleus was usually at the stage of metaphase I, rather than anaphase I as previously reported. At 26+/-2.5 minutes the meiotic process had entered the stage of metaphase II. The paternal and maternal pronuclei formed at 55+/-2.6 minutes, and they fused at 93+/-7.3 minutes. The mitotic division of the zygote was completed at 119+/-6.5 minutes.     

Tetraploid partial hydatidiform moles: two cases with a triple paternal contribution and a 92,XXXY karyotype

Summary In the course of a systematic study of cytogenetics, morphology, and clinical follow-up of hydatidiform moles we encountered two unusual cases of partial hydatidiform moles each with a 92,XXXY karyotype. Previously reported cases of tetraploidy, of 92,XXXX or 92,XXXY karyotype, resulted from a failure of the first mitotic division of a normal zygote. This is to our knowledge the first report of tetraploidy with XXXY sex chromosomes. Study of chromosomal heteromorphisms, isozymes, and restriction fragment length polymorphisms reveal that both present cases resulted from a combination of a haploid ovum with three haploid sets of paternal chromosomes either by the mechanism of trispermy (involving three separate haploid spermatozoa) or through dispermy (involving one haploid and one diploid sperm). Both cases resembled closely partial moles in their morphology; one gave a highly typical clinical picture while the other was recognized at an early voluntary abortion. Partial moles are ordinarily triploids of nearly always diandric constitution that evince focal villous swelling with cistern formation and focal trophoblastic hyperplasia. The findings here presented point to an association of molar phenotype with an excess of paternal over maternal haploid sets.     

Fertilization in Pinus Bungeana

Pinus bungeana is a species endemic to China and as yet its embryology has not been reported. The present paper deals with its process of fertilization in some details. 1. The development of the male gamete and the structure of the archegonium. The spermatogenous cell has already divided into two uniqual male gametes in the middle of May (in 1978, at Peking), about ten days before fertilization. Both sperms are spheroidal to ellipsoidal. The larger sperm is about 94 × 65 μm and the smaller one, about 72 × 58 μm in size. As the pollen tube approaches the archegonium the two sperms move toward the apex of the tube together with the remaining contents. Generally the larger sperm precedes the smaller one. The cytoplasmic contents also contain a sterile cell, 3—43×2—29 μm in size and a tube nuleus, 15—30 μm in diamter, besides the sperms. A mass of starch grains of more or less similar to sperm in size is also included in the contents of the pollen tube. Generally 3—4, even up to 7–8 pollen grains germinate normally within an ovule. Therefore, many sperms (up to 14—16) may be present on the same nucellus. The archegonium is elongato-ellipsoidal, about 870 ×500 μm in size. Arehegonia are single, 2—(3—5) in number, with 2 neck cells and a layer of jacket cells. The central cell divided in the middle of May and gave rise to the ventral canal cell and the egg. As the archegonium matures the cytoplasm becomes radiate fibrillae around the egg nucleus. The egg nucleus is large, 150—226 μm in diameter. One large nucleolus, 22—25 μm in diameter and sometimes up to 50; small nueleoli are present within the nucleus. 2. Fertilization Pollination takes place in the first week of May and fertilization will be effected from the end of May to the first week of June of next year. The interval between pollinatin and fertilization in P. bungeana is about thirteen months and the lapse of time is almost similar to most of the Pinus so far recorded. When the pollen tube contacts the archegonium through the neck cells all its contents are discharged into the egg cell. Usually the larger sperm fuses with the egg nucleus and the rest of the contents stays in the upper part of the egg cell. It is interesting to note that the nonfunctional second sperm also moves toward the egg nucleus and often divides by mitosis; and this phenomenon is not reported elsewhere. At the earlier stage of the fusion between male and female nuclei the male nucleoplasm is dense and finely granular while the female nucleoplasm is thin and coarsely granular, hence the boundary between them is very clear. The nuclear membranes of both nuclei persist for a long time. After the male nucleus sinks into the female nucleus completely, both nuclei begin to divide and enter into the prophase and then the metaphase simultaneously. By this time the paternal and maternal chromosome sets with their spindles still remain at certain distance from each other. Then the paternal chromosomes with their spindle move gradually toward the maternal ones. At first a multipolar common spindle appears as the maternal and paternal spindles with their chromosomes merge together. Finally a regular bipolar spindle is formed and both the maternal and paternal chromosomes become arranged on the equatorial plate. In the meantime, the process of fusion is complete and the zygote is at the stage of metaphase. At the moment the spindle looks greater in width than in length, being about 80×65—70 μm in size. 3. Supernumerary nuclei and sperms. The ventral canal cell degenerates soon after its formation. While the supernumerary sperms divide usually after their entrance into the egg cell. Therefore, the supernumerary nuclei probably derive directly from the smaller sperms or indirectly from mitoses of the larger ones Generally the nucleoplasm of the supernumerary nuclei is rather thin while the nucleoplasm of the undivided sperms is rather dense. This shows that the former is in the state of degeneration. The supernumerary nuclei of P. bungeana are as many as 7, their usual size being 43—58×32—43 μm. In the upper part of some egg cells there are still secondary smaller sperms about the size of 36 × 29 μm, Their volume is just about half of the usual smaller sperm. Probably they are derived from the division of the smaller sperms.     

The zygote and proembryo of alfalfa: quantitative,three-dimensional analysis and implications for biparental plastid inheritance

Summary Genetic studies have demonstrated biparental inheritance of plastids in alfalfa. The ratio of paternal to maternal plastids in the progeny varies according to the genotypes of the parents, which can be classified as strong or weak transmitters of plastids. Previous cytological investigations of generative cells and male gametes have provided no consistent explanation for plastid inheritance patterns among genotypes. However, plastids in the mature egg cells of a strong female genotype (6–4) were found to be more numerous and larger than in mature eggs of a weak female genotype (CUF-B), and the plastids in 6–4 eggs are positioned equally around the nucleus. In CUF-B, the majority of plastids are positioned below (toward the micropyle) the mid level of the nucleus, which is the future division plane of the zygote. Since only the apical portion of the zygote produces the embryo proper, plastids in the basal portion were predicted to become included in the suspensor cells and not be inherited. In the present study, we examined zygotes and a two-celled proembryo from a cross between CUF-B and a strong male genotype (301), a cross that results in over 90% of the progeny possessing paternal plastids only. Our results indicate that the distribution of plastids observed in the CUF-B egg cell is maintained through the first division of the zygote. Further, paternal plastids are similarly distributed; however, within the apical portion of the zygote and in the apical cell of the two-celled proembryo, the number of paternal plastids is typically much greater than the number of maternal plastids. These findings suggest that maternal and paternal plastid distribution within the zygote is a significant factor determining the inheritance of maternal and paternal plastids in alfalfa.     

Cytological evidence of biparental inheritance of plastids and mitochondria inPelargonium

Summary Based on the organelle differences between egg and sperm cells inPelargonium hortorum, the zygote, proembryo, and endosperm were examined under the transmission electron microscope. Plastids and mitochondria in the egg cell are significantly different from those of the sperm cell. Egg plastids are starch-containing and less electron dense. They appear circular, elliptical irregular elongate in sections. Sperm cell plastids are relatively electrondense, mostly cup-shaped or dumbbell and devoid of starch granules. Mitochondria of the egg cell are giant and mostly cup-shaped while sperm mitochondria are smaller and usually circular in section. Double fertilization is completed by 24 h after pollination and the pollen tube can be seen in the degenerated synergid. In the zygote, plastids and mitochondria from male and female gametes can be distinguished by their characteristic differences. Moreover, paternal and maternal organelles appear to be distributed at random in the zygote. Aside from the pollen tube and its released starch granules, there is no enucleated cytoplasmic body in the degenerated synergid. Two days after pollination, the zygote undergoes one transverse division to form a 2-celled proembryo which consists of one larger vacuolated basal cell and one smaller densely cytoplasmic apical cell. Paternal and maternal organelles can be detected in both cells of the proembryo and also in the endosperm at this stage. From these results, it can be concluded that plastids and mitochondria from both male and female gametes have been transmitted into the apical cell of the proembryo and most probably to the following generation.Abbreviations TEM transmission electron microscope - DAPI 4,6-diamidino-2-phenylindole - RFLP restriction fragment length polymorphism     

Chromosome constitution of in vitro segregated haploid and diploid cells of the mouse

The composition in segregated haploid sets of paternal and maternal chromosomes has been studied in order to verify whether their composition is uniparental of mixed, fixed or variable. Primary cultures where prepared using kidneys from hybrids of strains of Mus musculus in which the parental chromosomes are distinguishable; the maternal set consists of 20 teleocentric chromosomes, the paternal set of 9 metacentric chromosomes, derived by Robertsonian fusion and 2 telocentrics. Applying Seabright's banding technique, an analysis of segregated haploid and diploid cells, which have originated spontaneously through polyploidisation-segregation processes was carried out. It was concluded that the haploid sets have a variable composition of paternal and maternal chromosomes.     

gammaH2AX signalling during sperm chromatin remodelling in the mouse zygote

In the mouse, the paternal post-meiotic chromatin is assumed to be devoid of DNA repair after nuclear elongation and protamine-induced compaction. Hence, DNA lesions induced thereafter will have to be restored upon gamete fusion in the zygote. Misrepair of such lesions often results in chromosome type aberrations at the first cleavage division, suggesting that the repair event takes place prior to S-phase. During this stage of the zygotic cell cycle, the paternal chromatin transits from a protamine- to a nucleosome-based state. We addressed the question whether the canonical signalling pathway to DNA double strand breaks (DSBs), the phosphorylated form of histone H2AX (gammaH2AX) is active during chromatin restructuring of the male genetic complement in the zygote. Here, we describe the detailed characterization of gammaH2AX signalling in the early stages of zygotic development up to the appearance of the pronuclei. We have found the gammaH2AX signalling pathway to be already active during sperm chromatin remodelling after gamete fusion in a dose dependent manner, reflecting the amount of DSBs present in the sperm nucleus after in vivo male irradiation. Using DNA damaging compounds to induce lesions in the early zygote, differences in DSB sensitivity and gammaH2AX processing between paternal and maternal chromatin were found, suggesting differences in DNA repair capacity between the parental chromatin sets.     

离体受精作为技术平台在被子植物有性生殖研究中的应用

被子植物的离体受精10a前在玉米中已获得成功,尽管目前只在玉米获得完全成功和小麦获得部分成功,但离体受精技术的研究成果非常显著。目前离体受精技术已被用于其他的研究,如用分离的精细胞和卵细胞筛选配子细胞的特异基因和蛋白质：研究合子细胞被激活的机理：用不同种植物的精、卵细胞体外融合进行新的远缘杂交尝试;利用合子细胞易分裂和胚胎发生特征探索用其作为转基因研究的受体细胞等。以离体受精技术为基础在高等植物发育生物学和生殖生物学领域的基础研究和应用探索显示了巨大潜力。介绍了离体受精技术在被子植物有性生殖的研究成果和应用前景,为研究和利用被子植物有性生殖过程中的生殖细胞特征提供线索。     

The paternal genome in mouse zygotes is less sensitive to ENU mutagenesis than the maternal genome

The two parental genomes lie separate within the zygote and may be differentially affected by environmental influences. We have shown earlier (Russell et al., 1988) that the maternal genome within the mouse zygote is exquisitely sensitive to the induction of point mutations by N-ethyl-N-nitrosourea (ENU), and that the initial lesion probably occurs in one strand of the DNA. The present experiment measured specific-locus mutation induction in the paternal genome. Zygotes containing a multiple-recessive maternal genome (a; b; p cch; d se; s) and the corresponding wild-type alleles in the paternal one were exposed to 50 mg ENU/kg in vivo at one of two stages: the presumed times of sperm entry and early pronuclear stage. At weaning age, the resulting mice were examined for mutations at the marked loci as well as for other mutations producing externally visible phenotypes. At the marked loci, one possible mosaic (for b) was observed among 2113 classified offspring that had been treated with ENU as zygotes; this animal failed to transmit a mutation. By contrast, in the reciprocal cross (which tests the maternal genome) we had observed 8 specific-locus mutations (6 of them mosaics) among 1555 offspring that had received the same dose of ENU during sperm entry (and completion of oocyte meiosis II). In the present experiment, we also found one mutation at other loci (two at other loci in the reciprocal cross). The frequency of offspring with small white belly spots was significantly greater in the treated groups (3.5 and 1.9% at the earlier and later stage, respectively) than in the control (1.0%), the excess being almost entirely due to daughters. Genetic tests of a large number of such offspring failed to find a genetic cause. Instead, it appears that this phenotype may be influenced by factors in the intrauterine environment. It is concluded that shortly after sperm entry, the paternal genome of the zygote is less sensitive than the maternal one to the induction of mutations by ENU.     

Gamete fusion is required to block multiple pollen tubes from entering an Arabidopsis ovule

In double fertilization, a reproductive system unique to flowering plants, two immotile sperm are delivered to an ovule by a pollen tube. One sperm fuses with the egg to generate a zygote, the other with the central cell to produce endosperm. A mechanism preventing multiple pollen tubes from entering an ovule would ensure that only two sperm are delivered to female gametes. We use live-cell imaging and a novel mixed-pollination assay that can detect multiple pollen tubes and multiple sets of sperm within a single ovule to show that Arabidopsis efficiently prevents multiple pollen tubes from entering an ovule. However, when gamete-fusion defective hap2(gcs1) or duo1 sperm are delivered to ovules, as many as three additional pollen tubes are attracted. When gamete fusion fails, one of two pollen tube-attracting synergid cells persists, enabling the ovule to attract more pollen tubes for successful fertilization. This mechanism prevents the delivery of more than one pair of sperm to an ovule, provides a means of salvaging fertilization in ovules that have received defective sperm, and ensures maximum reproductive success by distributing pollen tubes to all ovules.     

A Brief Report on the Chromosome Number of Neodiplogaster pinicola and Panagrellus redivivoides (Nematoda: Rhabditida)

Both Neodiplogaster pinicola and Panagrellus redivivoides reproduce amphimictically, with XO type of sex determination. In N. pinicola, primary spermatocytes have six bivalent chromosomes and one univalent; after two meiotic divisions, sperm are produced with either six or seven chromosomes. In primary oocytes, with seven bivalents, meiosis is initiated by entrance of a sperm. After two meiotic divisions, three polar nuclei are produced, and egg and sperm pronuclei fuse. Cleavage begins after the egg is laid. Males have a 2n number of 13 chromosomes; females, 14. In P. redivivoides, primary spermatocytes have four bivalents and one univalent. After two meiotic divisions, spermatids are produced with either four or five well separated chromosomes. In primary oocytes, the first maturation division is initiated after penetration of a sperm; after two meiotic divisions, each egg has five chromosomes. Cleavage begins immediately after fusion of egg and sperm pronuclei, and embryonic development and hatching occur within the uterus. Males have a 2n chromosome number of 9; females, 10.     

EFFECTS OF MICROTUBULE INHIBITORS ON PRONUCLEAR MIGRATION AND EMBRYOGENESIS IN FUCUS DISTICHUS(PHAEOPHYTA)1

Pronuclear migration in Fucus distichus spp. edentatus (de la Pyl.) Powell is blocked by incubation of fertilized eggs in colchicine (1 mg/ml) and Nocodazole (2 μg/ ml). Rhizoids form prior to decondensation of the sperm chromatin in eggs in which pronuclear fusion is blocked. This occurs during continuous colchicine incubation as well as in eggs recovering from a short treatment with either drug following fertilization. During recovery of the cells, the sperm and egg chromosomes condense, and the sperm chromosomes migrate toward the egg pronucleus. The delay in migration following removal of colchicine is as much as 24 h and is even slower following removal of Nocodazole. The egg chromosomes form a metaphase plate in treated cells while the sperm chromosomes are still distant in the cytoplasm. This suggests that egg centrioles are important in the mitotic division of the zygote, not sperm centrioles. The effect of colchicine treatment on the mitotic plane and cytokinesis is also discussed.