Zygote Nucleus Research Articles

Temporospatial hierarchy and allele-specific expression of zygotic genome activation revealed by distant interspecific urochordate hybrids

Zygotic genome activation (ZGA) is a universal process in early embryogenesis of metazoan, when the quiescent zygotic nucleus initiates global transcription. However, the mechanisms related to massive genome activation and allele-specific expression (ASE) remain not well understood. Here, we develop hybrids from two deeply diverged (120 Mya) ascidian species to symmetrically document the dynamics of ZGA. We identify two coordinated ZGA waves represent early developmental and housekeeping gene reactivation, respectively. Single-cell RNA sequencing reveals that the major expression wave exhibits spatial heterogeneity and significantly correlates with cell fate. Moreover, allele-specific expression occurs in a species- rather than parent-related manner, demonstrating the divergence of cis-regulatory elements between the two species. These findings provide insights into ZGA in chordates.

Dynamics of mitochondrial distribution during development and asymmetric division of rice zygotes.

Mitochondria change their distribution from nuclear peripheral to uniformly distributed in cytoplasm during zygotic development of rice, and the mitochondria re-distribute around nucleus for even segregation into daughter cells. Mitochondria are highly dynamic organelles that actively move and change their localization along with actin filaments during the cell cycle. Studies of mitochondrial dynamics and distribution in plant cells have mainly been conducted on somatic cells, and our understanding about these aspects during the formation and development of zygotes remains limited. In this study, mitochondrial nucleoids of rice egg cells and zygotes were successfully stained by using N-aryl pyrido cyanine 3 (PC3), and their intracellular localization and distribution were demonstrated. Mitochondria in rice egg cells were small and coccoid in shape and were primarily distributed around the nucleus. Upon gamete fusion, the resulting zygotes showed mitochondrial dispersion and accumulation equivalent to those in rice egg cells until 8h after fusion (HAF). Around 12 HAF, the mitochondria started to disperse throughout the cytoplasm of the zygotes, and this dispersive distribution pattern continued until the zygotes entered the mitotic phase. At early prophase, the mitochondria redistributed from dispersive to densely accumulated around the nucleus, and during the metaphase and anaphase, the mitochondria were depleted from possible mitotic spindle region. Thereafter, during cell plate formation between daughter nuclei, the mitochondria distributed along the phragmoplast, where the new cell wall was formed. Finally, relatively equivalent amounts of mitochondria were detected in the apical and basal cells which were produced through asymmetric division of the zygotes. Further observation by treating the egg cell with latrunculin B revealed that the accumulation of mitochondria around the nuclear periphery in egg cells and early zygotes depended on the actin meshwork converging toward the egg or zygote nucleus.

AMPK Activity Contributes to G2 Arrest and DNA Damage Decrease via p53/p21 Pathways in Oxidatively Damaged Mouse Zygotes.

In zygotes, the capacity of G2/M checkpoint and DNA repair mechanisms to respond to DNA damage varies depending on different external stressors. In our previous studies, we found that mild oxidative stress induced a G2/M phase delay in mouse zygotes fertilized in vitro, due to the activation of the spindle assembly checkpoint. However, it is unclear whether the G2/M phase delay involves G2 arrest, triggered by activation of the G2/M checkpoint, and whether AMPK, a highly conserved cellular energy sensor, is involved in G2 arrest and DNA damage repair in mouse zygotes. Here, we found that mouse zygotes treated with 0.03 mM H2O2 at 7 h post-insemination (G1 phase), went into G2 arrest in the first cleavage. Furthermore, phosphorylated H2AX, a specific DNA damage and repair marker, can be detected since the early S phase. We also observed that oxidative stress induced phosphorylation and activation of AMPK. Oxidative stress-activated AMPK first localized in the cytoplasm of the mouse zygotes in the late G1 phase and then translocated to the nucleus from the early S phase. Overall, most of the activated AMPK accumulated in the nuclei of mouse zygotes arrested in the G2 phase. Inhibition of AMPK activity with Compound C and SBI-0206965 abolished oxidative stress-induced G2 arrest, increased the activity of CDK1, and decreased the induction of cell cycle regulatory proteins p53 and p21. Moreover, bypassing G2 arrest after AMPK inhibition aggravated oxidative stress-induced DNA damage at M phase, increased the apoptotic rate of blastocysts, and reduced the formation rate of 4-cell embryos and blastocysts. Our results suggest the G2/M checkpoint and DNA repair mechanisms are operative in coping with mild oxidative stress-induced DNA damage. Further, AMPK activation plays a vital role in the regulation of the oxidative stress-induced G2 arrest through the inhibition of CDK1 activity via p53/p21 pathways, thereby facilitating the repair of DNA damage and the development and survival of oxidative stress-damaged embryos. Our study provides insights into the molecular mechanisms underlying oxidative-stress induced embryonic developmental arrest, which is crucial for the development of novel strategies to ensure viable embryo generation.

Evolution, Initiation, and Diversity in Early Plant Embryogenesis.

During embryogenesis in plants, cell identities are specified de novo, starting from a single cell. By combining imaging, genomic profiling, and genetics, principles of early plant development have been unraveled in the dicotyledonous plant Arabidopsis. A central emerging question, however, is how well zygotic embryogenesis in Arabidopsis reflects homologous processes in other plant species, including early diverging, non-flowering, and non-seed plants. Here, we consider plant embryogenesis with an emphasis on its evolutionary history, the diverse modes of its initiation, and the concepts in pattern formation among morphologically distinct plant groups. Furthermore, we explore challenges and future directions in plant embryogenesis research.

Physiological polyspermy: Selection of a sperm nucleus for the development of diploid genomes in amphibians.

The union between a sperm and an egg nucleus in egg fertilization is necessary to mix genetic materials to create a new diploid genome for the next generation. In most animals, only one sperm is incorporated into the egg (monospermy), but several animals exhibit physiological polyspermy in which several sperms enter the egg during normal fertilization. However, only one sperm nucleus forms the zygote nucleus with the egg nucleus, even in a polyspermic egg. The cellular and molecular mechanisms involved in the selection of sperm nuclei in the egg cytoplasm have been well investigated in urodele amphibians. The principal sperm nucleus develops a larger sperm aster and contacts the egg nucleus to form a zygote nucleus, whereas other accessory sperm nuclei are unable to approach the egg nucleus. The diploid zygote nucleus induces cleavage and participates in embryonic development, whereas the accessory sperm nuclei undergo pyknosis and degenerate. We propose several models to account for the mechanisms of the selection of one sperm nucleus and the degeneration of accessory sperm nuclei. The roles of physiological polyspermy in animal reproduction are discussed by comparison with other polyspermic species.

Nuclear (Bio)physics in the Embryo

Deneke etal. (2019) discover that dynamic interactions of cell cycle and actomyosin contractility systems synchronize nuclear cleavages, generating a cytoplasmic flow that results in a spatially uniform distribution of zygotic nuclei in the early Drosophila embryo. This work underscores the importance of self-organizing mechanisms before the onset of zygotic transcription.

Twisted Tales: Insights into Genome Diversity of Ciliates Using Single-Cell 'Omics.

The emergence of robust single-cell ‘omics techniques enables studies of uncultivable species, allowing for the (re)discovery of diverse genomic features. In this study, we combine single-cell genomics and transcriptomics to explore genome evolution in ciliates (a > 1 Gy old clade). Analysis of the data resulting from these single-cell ‘omics approaches show: 1) the description of the ciliates in the class Karyorelictea as “primitive” is inaccurate because their somatic macronuclei contain loci of varying copy number (i.e., they have been processed by genome rearrangements from the zygotic nucleus); 2) gene-sized somatic chromosomes exist in the class Litostomatea, consistent with Balbiani’s (1890) observation of giant chromosomes in this lineage; and 3) gene scrambling exists in the underexplored Postciliodesmatophora (the classes Heterotrichea and Karyorelictea, abbreviated here as the Po-clade), one of two major clades of ciliates. Together these data highlight the complex evolutionary patterns underlying germline genome architectures in ciliates and provide a basis for further exploration of principles of genome evolution in diverse microbial lineages.

Insights into an Extensively Fragmented Eukaryotic Genome: De Novo Genome Sequencing of the Multinuclear Ciliate Uroleptopsis citrina.

Ciliated protists are a large group of single-celled eukaryotes with separate germline and somatic nuclei in each cell. The somatic genome is developed from the zygotic nucleus through a series of chromosomal rearrangements, including fragmentation, DNA elimination, de novo telomere addition, and DNA amplification. This unique feature makes them perfect models for research in genome biology and evolution. However, genomic research of ciliates has been limited to a few species, owing to problems with DNA contamination and obstacles in cultivation. Here, we introduce a method combining telomere-primer PCR amplification and high-throughput sequencing, which can reduce DNA contamination and obtain genomic data efficiently. Based on this method, we report a draft somatic genome of a multimacronuclear ciliate, Uroleptopsis citrina. 1) The telomeric sequence in U. citrina is confirmed to be C4A4C4A4C4 by directly blunt-end cloning. 2) Genomic analysis of the resulting chromosomes shows a “one-gene one-chromosome” pattern, with a small number of multiple-gene chromosomes. 3) Amino acid usage is analyzed, and reassignment of stop codons is confirmed. 4) Chromosomal analysis shows an obvious asymmetrical GC skew and high bias between A and T in the subtelomeric regions of the sense-strand, with the detection of an 11-bp high AT motif region in the 3′ subtelomeric region. 5) The subtelomeric sequence also has an obvious 40 nt strand oscillation of nucleotide ratio. 6) In the 5′ subtelomeric region of the coding strand, the distribution of potential TATA-box regions is illustrated, which accumulate between 30 and 50 nt. This work provides a valuable reference for genomic research and furthers our understanding of the dynamic nature of unicellular eukaryotic genomes.

Delayed Encounter of Parental Genomes Can Lead to Aneuploidy in Saccharomyces cerevisiae.

We have investigated an extreme deviation from the norm of genome unification that occurs during mating in the yeast, Saccharomyces cerevisiae This deviation is encountered when yeast that carry a mutation of the spindle pole body protein, Kar1, are mated with wildtype cells. In this case, nuclear fusion is delayed and the genotypes of a fraction of zygotic progeny suggest that chromosomes have "transferred" between the parental nuclei in zygotes. This classic, yet bizarre, occurrence is routinely used to generate aneuploid (disomic) yeast. [kar1×wt] zygotes, like [wt×wt] zygotes, initially have a single spindle pole body per nucleus. Unlike [wt×wt] zygotes, in [kar1×wt] zygotes, the number of spindle pole bodies per nucleus then can increase before nuclear fusion. When such nuclei fuse, the spindle pole bodies do not coalesce efficiently, and subsets of spindle pole bodies and centromeres can enter buds. The genotypes of corresponding biparental progeny show evidence of extensive haplotype-biased chromosome loss, and can also include heterotypic chromosomal markers. They thus allow rationalization of chromosome "transfer" as being due to an unanticipated yet plausible mechanism. Perturbation of the unification of genomes likely contributes to infertility in other organisms.

Cell cycle in egg cell and its progression during zygotic development in rice.

Rice egg is arrested at G1 phase probably by OsKRP2. After fusion with sperm, karyogamy, OsWEE1-mediated parental DNA integrity in zygote nucleus, zygote progresses cell cycle to produce two-celled embryo. In angiosperms, female and male gametes exist in gametophytes after the complementation of meiosis and the progression of nuclear/cell division of the haploid cell. Within the embryo sac, the egg cell is specially differentiated for fertilization and subsequent embryogenesis, and cellular programs for embryonic development, such as restarting the cell cycle and de novo gene expression, are halted. There is only limited knowledge about how the cell cycle in egg cells restarts toward zygotic division, although the conversion of the cell cycle from a quiescent and arrested state to an active state is the most evident transition of cell status from egg cell to zygote. This is partly due to the difficulty in direct access and analysis of egg cells, zygotes and early embryos, which are deeply embedded in ovaries. In this study, precise relative DNA amounts in the nuclei of egg cells, developing zygotes and cells of early embryos were measured, and the cell cycle of a rice egg cell was estimated as the G1 phase with a 1C DNA level. In addition, increases in DNA content in zygote nuclei via karyogamy and DNA replication were also detectable according to progression of the cell cycle. In addition, expression profiles for cell cycle-related genes in egg cells and zygotes were also addressed, and it was suggested that OsKRP2 and OsWEE1 function in the inhibition of cell cycle progression in egg cells and in checkpoint of parental DNA integrity in zygote nucleus, respectively.

Development of polyspermic zygote and possible contribution of polyspermy to polyploid formation in angiosperms.

Fertilization is a general feature of eukaryotic uni- and multicellular organisms to restore a diploid genome from female and male gamete haploid genomes. In angiosperms, polyploidization is a common phenomenon, and polyploidy would have played a major role in the long-term diversification and evolutionary success of plants. As for the mechanism of formation of autotetraploid plants, the triploid-bridge pathway, crossing between triploid and diploid plants, is considered as a major pathway. For the emergence of triploid plants, fusion of an unreduced gamete with a reduced gamete is generally accepted. In addition, the possibility of polyspermy has been proposed for maize, wheat and some orchids, although it has been regarded as an uncommon mechanism of triploid formation. One of the reasons why polyspermy is regarded as uncommon is because it is difficult to reproduce the polyspermy situation in zygotes and to analyze the developmental profiles of polyspermic triploid zygotes. Recently, polyspermic rice zygotes were successfully produced by electric fusion of an egg cell with two sperm cells, and their developmental profiles were monitored. Two sperm nuclei and an egg nucleus fused into a zygotic nucleus in the polyspermic zygote, and the triploid zygote divided into a two-celled embryo via mitotic division with a typical bipolar microtubule spindle. The two-celled proembryos further developed and regenerated into triploid plants. These suggest that polyspermic plant zygotes have the potential to form triploid embryos, and that polyspermy in angiosperms might be a pathway for the formation of triploid plants.

Transgenerational function of Tetrahymena Piwi protein Twi8p at distinctive noncoding RNA loci.

Transgenerational transmission of genome-regulatory epigenetic information can determine phenotypes in the progeny of sexual reproduction. Sequence specificity of transgenerational regulation derives from small RNAs assembled into Piwi-protein complexes. Known targets of transgenerational regulation are primarily transposons and transposon-derived sequences. Here, we extend the scope of Piwi-mediated transgenerational regulation to include unique noncoding RNA loci. Ciliates such as Tetrahymena have a phenotypically silent germline micronucleus and an expressed somatic macronucleus, which is differentiated anew from a germline genome copy in sexual reproduction. We show that the nuclear-localized Tetrahymena Piwi protein Twi8p shuttles from parental to zygotic macronuclei. Genetic elimination of Twi8p has no phenotype for cells in asexual growth. On the other hand, cells lacking Twi8p arrest in sexual reproduction with zygotic nuclei that retain the germline genome structure, without the DNA elimination and fragmentation required to generate a functional macronucleus. Twi8p-bound small RNAs originate from long-noncoding RNAs with a terminal hairpin, which become detectable in the absence of Twi8p. Curiously, the loci that generate Twi8p-bound small RNAs are essential for asexual cell growth, even though Twi8 RNPs are essential only in sexual reproduction. Our findings suggest the model that Twi8 RNPs act on silent germline chromosomes to permit their conversion to expressed macronuclear chromosomes. Overall this work reveals that a Piwi protein carrying small RNAs from long-noncoding RNA loci has transgenerational function in establishing zygotic nucleus competence for gene expression.

Parthenogenesis in Insects: The Centriole Renaissance.

Building a new organism usually requires the contribution of two differently shaped haploid cells, the male and female gametes, each providing its genetic material to restore diploidy of the new born zygote. The successful execution of this process requires defined sequential steps that must be completed in space and time. Otherwise, development fails. Relevant among the earlier steps are pronuclear migration and formation of the first mitotic spindle that promote the mixing of parental chromosomes and the formation of the zygotic nucleus. A complex microtubule network ensures the proper execution of these processes. Instrumental to microtubule organization and bipolar spindle assembly is a distinct non-membranous organelle, the centrosome. Centrosome inheritance during fertilization is biparental, since both gametes provide essential components to build a functional centrosome. This model does not explain, however, centrosome formation during parthenogenetic development, a special mode of sexual reproduction in which the unfertilized egg develops without the contribution of the male gamete. Moreover, whereas fertilization is a relevant example in which the cells actively check the presence of only one centrosome, to avoid multipolar spindle formation, the development of parthenogenetic eggs is ensured, at least in insects, by the de novo assembly of multiple centrosomes.Here, we will focus our attention on the assembly of functional centrosomes following fertilization and during parthenogenetic development in insects. Parthenogenetic development in which unfertilized eggs are naturally depleted of centrosomes would provide a useful experimental system to investigate centriole assembly and duplication together with centrosome formation and maturation.

Formation of triploid plants via possible polyspermy

ABSTRACTPolyploidization is a common phenomenon in angiosperms, and polyploidy has played a major role in the long-term diversification and evolutionary success of plants. Triploid plants are considered as the intermediate stage in the formation of stable autotetraploid plants, and this pathway of tetraploid formation is known as the triploid bridge. As for the mechanism of triploid formation among diploid populations, fusion of an unreduced gamete with a reduced gamete is generally accepted. In addition, the possibility of polyspermy has been proposed for maize, wheat and some orchids, although it has been regarded as an uncommon mechanism of polyploid formation. One of the reasons why polyspermy is regarded as uncommon is because it is difficult to reproduce the polyspermy situation in zygotes and to analyze the developmental profiles of polyspermic zygotes. In the study, we produced polyspermic rice zygotes by electric fusion of an egg cell with two sperm cells and monitored their developmental profiles. The two sperm nuclei and the egg nucleus fused into a zygotic nucleus in the polyspermic zygote, and the triploid zygote divided into a two-celled embryo via mitotic division with a typical bipolar microtubule spindle. The two-celled proembryos developed and regenerated into triploid plants. These results suggest that polyspermic plant zygotes have the potential to form triploid embryos, and that polyspermy in angiosperms might be a pathway for the formation of triploid plants.

Lolal Is an Evolutionarily New Epigenetic Regulator of dpp Transcription during Dorsal-Ventral Axis Formation.

Secreted ligands in the Dpp/BMP family drive dorsal–ventral (D/V) axis formation in all Bilaterian species. However, maternal factors regulating Dpp/BMP transcription in this process are largely unknown. We identified the BTB domain protein longitudinals lacking-like (lolal) as a modifier of decapentaplegic (dpp) mutations. We show that Lolal is evolutionarily related to the Trithorax group of chromatin regulators and that lolal interacts genetically with the epigenetic factor Trithorax-like during Dpp D/V signaling. Maternally driven LolalHA is found in oocytes and translocates to zygotic nuclei prior to the point at which dpp transcription begins. lolal maternal and zygotic mutant embryos display significant reductions in dpp, pMad, and zerknullt expression, but they are never absent. The data suggest that lolal is required to maintain dpp transcription during D/V patterning. Phylogenetic data revealed that lolal is an evolutionarily new gene present only in insects and crustaceans. We conclude that Lolal is the first maternal protein identified with a role in dpp D/V transcriptional maintenance, that Lolal and the epigenetic protein Trithorax-like are essential for Dpp D/V signaling and that the architecture of the Dpp D/V pathway evolved in the arthropod lineage after the separation from vertebrates via the incorporation of new genes such as lolal.

Development of Polyspermic Rice Zygotes.

Fertilization is a general feature of eukaryotic uni- and multicellular organisms to restore a diploid genome from female and male gamete haploid genomes. In most animals and fucoid algae, polyspermy block occurs at the plasmogamy step. Because the polyspermy barrier in animals and in fucoid algae is incomplete, polyspermic zygotes are generated by multiple fertilization events. However, these polyspermic zygotes with extra centrioles from multiple sperms show aberrant nuclear and cell division. In angiosperms, polyspermy block functions in the egg cell and the central cell to promote faithful double fertilization, although the mechanism of polyspermy block remains unclear. In contrast to the case in animals and fucoid algae, polyspermic zygotes formed in angiosperms are not expected to die because angiosperms lack centrosomes. However, there have been no reports on the developmental profiles of polyspermic zygotes at cellular level in angiosperms. In this study, we produced polyspermic rice zygotes by electric fusion of an egg cell with two sperm cells, and monitored their developmental profiles. Two sperm nuclei and an egg nucleus fused into a zygotic nucleus, and the triploid zygote divided into a two-celled embryo via mitotic division with a typical bipolar microtubule spindle, as observed during mitosis of a diploid zygote. The two-celled proembryos further developed and regenerated into triploid plants. These findings suggest that polyspermic plant zygotes have the potential to form triploid embryos. Polyspermy in angiosperms might be a pathway for the formation of triploid plants, which can contribute significantly to the formation of autopolyploids.

Archegonium and fertilization in Coniferopsida

This paper reviews studies on the origin and development of the Coniferopsida’s archegonium, illustrates the relationship between the archegonium and fertilization, and discusses the molecular regulation of archegonium development. This paper reviews studies on the origin and development of the Coniferopsida’s archegonium and discusses the characteristics of the archegonial cells and the molecular regulation of archegonium development. There are some main conclusions which could be drawn from this review. (1) It enables the precise position of the archegonium in the ovule to be determined as well as its structural features. (2) The functions of the neck cells in Coniferopsida are that they may serve as channels for sperms to enter the archegonia and lead the growth of pollen tubes. (3) The proteid vacuoles in egg cell may provide nutrition for fertilization and lead to the paternal inheritance of plastids in some Coniferopsida. (4) The uniparental transmission of plastids results from “the active digestion of male organelle nuclei (genomes) in young zygotes” in almost all Coniferopsida. (5) The studies on the molecular regulation of the archegonium and fertilization in Coniferopsida are still very limited and that is the direction for further research. In summary, the review provides a theoretical basis for further study on the developmental and reproductive biology of the Coniferopsida.

TFIIS-Dependent Non-coding Transcription Regulates Developmental Genome Rearrangements.

Because of their nuclear dimorphism, ciliates provide a unique opportunity to study the role of non-coding RNAs (ncRNAs) in the communication between germline and somatic lineages. In these unicellular eukaryotes, a new somatic nucleus develops at each sexual cycle from a copy of the zygotic (germline) nucleus, while the old somatic nucleus degenerates. In the ciliate Paramecium tetraurelia, the genome is massively rearranged during this process through the reproducible elimination of repeated sequences and the precise excision of over 45,000 short, single-copy Internal Eliminated Sequences (IESs). Different types of ncRNAs resulting from genome-wide transcription were shown to be involved in the epigenetic regulation of genome rearrangements. To understand how ncRNAs are produced from the entire genome, we have focused on a homolog of the TFIIS elongation factor, which regulates RNA polymerase II transcriptional pausing. Six TFIIS-paralogs, representing four distinct families, can be found in P. tetraurelia genome. Using RNA interference, we showed that TFIIS4, which encodes a development-specific TFIIS protein, is essential for the formation of a functional somatic genome. Molecular analyses and high-throughput DNA sequencing upon TFIIS4 RNAi demonstrated that TFIIS4 is involved in all kinds of genome rearrangements, including excision of ~48% of IESs. Localization of a GFP-TFIIS4 fusion revealed that TFIIS4 appears specifically in the new somatic nucleus at an early developmental stage, before IES excision. RT-PCR experiments showed that TFIIS4 is necessary for the synthesis of IES-containing non-coding transcripts. We propose that these IES+ transcripts originate from the developing somatic nucleus and serve as pairing substrates for germline-specific short RNAs that target elimination of their homologous sequences. Our study, therefore, connects the onset of zygotic non coding transcription to the control of genome plasticity in Paramecium, and establishes for the first time a specific role of TFIIS in non-coding transcription in eukaryotes.

The cytological changes of sperm and oocyte nuclei during fertilization in Phascolosoma esculenta (Sipuncula: Phascolosomatidea)

In this study, we investigated fertilization cytology in Phascolosoma esculenta. The sperm binds to the oocyte envelope in a random position at the point of initial contact. An acrosomal reaction then occurs and the acrosomal filament penetrates the oocyte envelope. The apex of the acrosomal filament contacts with the plasma membrane of the oocyte at the point where the fertilization cone is formed. The fertilization cone then retracts, towing the acrosomal filament back and drawing the sperm into the oocyte. Subsequently, the pores on the oocyte envelope are blocked and filled with fibrin and colloid. After the oocyte is activated by sperm, the first and second meiosis processes start sequentially, followed by the release of the first and second polar bodies. After the second polar body is extruded, the haploid female nucleus rapidly reorganizes to form a female pronucleus. In comparison, the sperm nucleus begins to decondense and dilate after the first polar body is released. The male pronucleus is not formed until the end of the second meiosis. The male and female pronuclei migrate to the centre of the oosperm and are incorporated into the zygote nucleus and then the first cleavage occurs. A two cell embryo is then formed by plasma membrane invagination. The cytological changes of sperm and oocyte nuclei during sperm entry into the oocyte and the fertilization process in P. esculenta are characterized by: (1) the mature oocyte with fertilization ability being in the metaphase of the first meiosis; (2) sperm is drawn into the oocyte by the acrosomal filament and fertilization cone; (3) some abnormal phenomena such as polyspermy, multiple pronuclei and multipolar spindle; and (4) the male pronucleus is formed earlier than the female pronucleus, at which point they will fuse to form a zygote nucleus.

The expression of an immune-related phenoloxidase gene is modulated in Ciona intestinalis ovary, test cells, embryos and larva.

Two distinct Ciona intestinalis phenoloxidases (CinPO1, 2) had previously been cloned and sequenced. The CinPO2 is involved in innate immunity and is expressed by inflammatory hemocytes that populate the tunic and pharynx vessels as a response to LPS inoculation. In situ hybridization and immunohistochemistry assays on histological section, showed that the expression of this gene and the produced protein are shared with oogenesis, embryogenesis and larval morphogenesis. Intriguingly, upregulation of gene transcription was found in the test cell layer that envelopes the ovary follicle, ovulated egg, and gastrula, as well as it was modulated in the zygotic nucleus of outer balstomers of 32-cell embryo, neurula presumptive epidermis tissue and larval mesenchyme. The anti-CinPO2 antibodies, specific for adult inflammatory cells, recognize epitopes in the cytoplasm of ovarian oocytes, ovulated eggs, development stages and larval mesenchyme. The overall findings disclose the precocious activation of the CinPO2 immunity-related gene, and show a developmentally programmed expression of this phenoloxidase. Furthermore, these findings support the multifunctional roles of immunity-related genes and allows us to explore new perspectives on ascidian development and immunity.