Female Gametogenesis Research Articles

SLOW WALKER3, Encoding a Putative DEAD‐box RNA Helicase, is Essential for Female Gametogenesis in Arabidopsis

RNA helicases are adenosine tri-phosphatases that unwind the secondary structures of RNAs and are required in almost any aspect of RNA metabolism. They are highly conserved from prokaryotic to eukaryotic organisms. However, their precise roles in plant physiology and development remain to be clarified. Here we report that the mutation in the gene SLOW WALKER3 (SWA3) results in the slow and retarded progression of mitosis during megagametogenesis in Arabidopsis. SWA3 is a putative RNA helicase of the DEAD-box subfamily. Mutant megagametophyte development is arrested at four- or eight-nucleate stages, furthermore, one of the synergids in about half of the mutant embryo sacs displays abnormal polarity, with its nucleus locating at the chalazal end, instead of the micropylar end in the wild-type. Transmission of the mutation through female gametophytes is severely reduced in swa3. However, a small portion of mutant embryo sacs are able to develop into mature and functional female gametophytes when pollination was postponed. The SWA3 in Arabidopsis is a homolog of Dbp8 in yeast. Dbp8 interacts with Efs2 and is essential for biogenesis of 18S rRNA in yeast. Our data suggest that SWA3 may form a complex with AtEfs2 and take roles in ribosomal biogenesis as RNA helicase during megagametogenesis in Arabidopsis.

DNA methylation reprogramming during plant sexual reproduction?

Chromatin modifications including histone marks and DNA methylation restrict the transcriptional repertoire and participate in cell fate establishment. Conservation of modified chromatin states through cell division and their inheritance through meiosis create mitotic and trans-generational forms of epigenetic memory, respectively. This lies in apparent contradiction with the requirement to reset cell-fate instructive chromatin states between generations. Although DNA methylation is reset in mammals, its resetting in plants remains controversial, and several lines of evidence support trans-generational inheritance of DNA methylation. Based on recent reports we propose that DNA demethylation during female gametogenesis is followed by DNA remethylation during early embryo development. We propose that this reprogramming event is achieved through interplay between active and passive mechanisms that involve both DNA demethylation and de novo DNA methylation.

The cytohistological basis of apospory in Hypericum perforatum L.

St. John's wort (Hypericum perforatum L., 2n = 4x = 32) is a medicinal plant that produces pharmaceutically important metabolites with antidepressive, anticancer and antiviral activities. It is also regarded as a serious weed in many countries. H. perforatum is furthermore an attractive model system for the study of apomixis. Natural populations of H. perforatum are predominantly composed of tetraploid individuals, although diploids and hexaploids are known to occur. It has been demonstrated that while diploids are sexual, polyploids are facultative apomictic whereby a single individual can produce both sexual and apomictic seeds. Despite our increasing understanding of gamete formation in sexually reproducing species, relatively little is known regarding the cytological basis of reproduction in H. perforatum. Here, we have studied embryo sac formation and the genetic constitution of seeds by means of staining-clearing of ovules/ovaries, DIC microscopy and flow cytometric seed screening (FCSS) of embryo and endosperm DNA contents. Comparisons of female sporogenesis and gametogenesis between sexual and apomictic accessions have enabled the identification of major phenotypic differences in embryo sac formation, in addition to complex fertilization scenarios entailing reduced and unreduced male and female gametes. These data provide new insights into the production of aposporous seeds in H. perforatum, and complement ongoing population genetic, genomic and transcriptomic studies.

Sporogenesis and gametogenesis in Agapanthus praecox Willd. orientalis (Leighton) Leighton and their systematic implications

Micro- and megasporogenesis as well as male and female gametogenesis in genus Agapanthus are reported for the first time. The results are as follows: The anther wall, whose development is of dicotyledonous type, consists of four layers: the epidermis, the endothecium, one or two middle layers, and the secretory tapetum. Fibrous thickenings are developed in the endothecium when shed. Successive cytokinesis during microsporogenesis results in a decussate tetrad of microspores. Ellipsoidal pollen grains are 2-celled when anthers dehisce, with monosulcate extending to polar area; pollen ornamentation is pitted and reticulate. Ovary is superior and trilocular, with axial placenta. The ovule is anatropous, bitegmic, and tenuinucellate. The archesporial cell below nucellus epidermis functions directly as the megasporocyte. Successive cytokinesis in the megasporocyte usually produces a T-shape tetrad, and the chalazal megaspore of the tetrad develops into a Polygonum-type embryo sac. The systematic significance of embryological characters of Agapanthus is discussed.

Molecular profiling of human oocytes after vitrification strongly suggests that they are biologically comparable with freshly isolated gametes

To assess the effects of vitrification on the biomolecular profile of oocytes, we analyzed through real-time reverse transcriptase-polymerase chain reaction eight genes encoding critically important proteins for embryo development and compared this partial transcriptome with that of freshly collected gametes isolated from the same women. The comparison of the molecular profiles demonstrated that our vitrification protocol does not alter the biomolecular quality of oocytes: in fact, between the two groups we found the absence of statistically significant variations. Accordingly, this cryopreservation technique might be helpful in preserving women's fertility.

Repressive and active histone methylation mark distinct promoters in human and mouse spermatozoa

In higher eukaryotes, histone methylation is involved in maintaining cellular identity during somatic development. As most nucleosomes are replaced by protamines during spermatogenesis, it is unclear whether histone modifications function in paternal transmission of epigenetic information. Here we show that two modifications important for Trithorax- and Polycomb-mediated gene regulation have methylation-specific distributions at regulatory regions in human spermatozoa. Histone H3 Lys4 dimethylation (H3K4me2) marks genes that are relevant in spermatogenesis and cellular homeostasis. In contrast, histone H3 Lys27 trimethylation (H3K27me3) marks developmental regulators in sperm, as in somatic cells. However, nucleosomes are only moderately retained at regulatory regions in human sperm. Nonetheless, genes with extensive H3K27me3 coverage around transcriptional start sites in particular tend not to be expressed during male and female gametogenesis or in preimplantation embryos. Promoters of orthologous genes are similarly modified in mouse spermatozoa. These data are compatible with a role for Polycomb in repressing somatic determinants across generations, potentially in a variegating manner.

Annual reproductive cycle of the oyster, Saccostrea kegaki (Torigoe & Inaba 1981) on the southern coast of Jeju island, Korea

Gametogenesis of the oyster Saccostrea kegaki (Torigoe & Inaba, 1981) on the southern coast of Jeju (Cheju) Island, Korea was assessed monthly (March 2001 to February 2002). Gametogenesis in females and males commenced in April when the water temperature reached 16.5°C. Ripe gametes were observed from June to September in both sexes. Partially spawned oysters were evident from July to October when the water temperature ranged 24.6–26.7°C. Most oysters collected in July and August exhibited partially empty follicles with large eggs (>35 μm in diameter), suggesting that these two months are the major spawning periods. From November to March, most female oysters were in resting stage exhibiting no gametes in the follicles. In contrast, spawning or spent male oysters could be seen during November and January. Monthly mean oocyte diameters ranged from 10.3 ± 1.7 μm in April (early developing stage) to 36.9 ± 5.7 μm in August (ripe stage) and the mature oocyte was comparatively smaller than the size found in other oysters. The relatively slow rate of gametogenesis observed in S. kegaki off southern Jeju could be in part explained by low level of food in the water column rather than by water temperature.

A DEAD-Box Protein, AtRH36, is Essential for Female Gametophyte Development and is Involved in rRNA Biogenesis in Arabidopsis

DEAD-box RNA helicases are involved in RNA metabolism, including pre-mRNA splicing, ribosome biogenesis, RNA decay and gene expression. In this study, we identified a homolog of the RH36 gene, AtRH36, which encodes a DEAD-box protein in Arabidopsis thaliana. The gene was expressed ubiquitously throughout the plant. The AtRH36 fused to green fluorescent protein was localized in the nucleus. Homozygosity for the Arabidopsis atrh36 mutants, atrh36-1 and atrh36-2, could not be obtained. Progeny of selfed Arabidopsis atrh36 heterozygote plants were obtained at a heterozygote to wild-type ratio of 1 : 1, which suggested that the AtRH36 gene was involved in gametogenesis. Therefore, we performed a reciprocal cross to determine whether AtRH36 was involved in female gametophyte development. Female gametogenesis was delayed in atrh36-1, and asynchronous development of the female gametophytes was found within a single pistil. Knock-down of AtRH36 gave a pleiotropic phenotype and led to the accumulation of unprocessed 18S pre-rRNA. These results suggest that AtRH36 is essential for mitotic division during female gametogenesis and plays an important role in rRNA biogenesis in Arabidopsis.

Mutations in the Arabidopsis Nuclear-Encoded Mitochondrial Phage-Type RNA Polymerase Gene RPOTm Led to Defects in Pollen Tube Growth, Female Gametogenesis and Embryogenesis

The mitochondrial genes in Arabidopsis thaliana are transcribed by a small family of nuclear-encoded T3/T7 phage-type RNA polymerases (RPOTs). At least two nuclear-encoded RPOTs (RPOTm and RPOTmp) are located in mitochondria in A. thaliana. Their genetic roles are largely unknown. Here we report the characterization of novel mutations in the A. thaliana RPOTm gene. The mutations did not affect pollen formation, but significantly retarded the growth of the rpoTm mutant pollen tubes and had an impact on the fusion of the polar nuclei in the rpoTm mutant embryo sacs. Moreover, development of the rpoTm/- mutant embryo was arrested at the globular stage. The rpoTm rpoTmp double mutation could enhance the rpoTm mutant phenotype. Expression of RPOTmp under control of the RPOTm promoter could not complement the phenotype of the rpoTm mutations. All these data indicate that RPOTm is important for normal pollen tube growth, female gametogenesis and embryo development, and has distinct genetic and molecular roles in plant development, which cannot be replaced by RPOTmp.

Mitochondrie et maturation ovocytaire

Mitochondria play a central role in cellular energy metabolism. Mitochondrial genome (mtDNA) is specific with a maternal transmission. Defects of mtDNA, both quantitative (depletion) but also qualitative (mutation) were observed in patients with reproductive disorders, suggesting that a mitochondrial deficit could cause a failure of oocyte maturation. In our experience, some mtDNA point mutations would be subject to negative selection during female gametogenesis, with a threshold rate depending on the mutation. Below this threshold, the presence of mtDNA mutations would not impact oocyte maturation nor early embryonic development, as evidenced by the large number of oocytes and embryos carrying high mutation rates in our series.

One Egg at a Time

Silencing of transposable elements by ARGONAUTE 9 is necessary to specify a single female gamete in Arabidopsis .

Control of female gamete formation by a small RNA pathway in Arabidopsis

In the ovules of most sexual flowering plants female gametogenesis is initiated from a single surviving gametic cell, the functional megaspore, formed after meiosis of the somatically derived megaspore mother cell (MMC). Because some mutants and certain sexual species exhibit more than one MMC, and many others are able to form gametes without meiosis (by apomixis), it has been suggested that somatic cells in the ovule are competent to respond to a local signal likely to have an important function in determination. Here we show that the Arabidopsis protein ARGONAUTE 9 (AGO9) controls female gamete formation by restricting the specification of gametophyte precursors in a dosage-dependent, non-cell-autonomous manner. Mutations in AGO9 lead to the differentiation of multiple gametic cells that are able to initiate gametogenesis. The AGO9 protein is not expressed in the gamete lineage; instead, it is expressed in cytoplasmic foci of somatic companion cells. Mutations in SUPPRESSOR OF GENE SILENCING 3 and RNA-DEPENDENT RNA POLYMERASE 6 exhibit an identical defect to ago9 mutants, indicating that the movement of small RNA (sRNAs) silencing out of somatic companion cells is necessary for controlling the specification of gametic cells. AGO9 preferentially interacts with 24-nucleotide sRNAs derived from transposable elements (TEs), and its activity is necessary to silence TEs in female gametes and their accessory cells. Our results show that AGO9-dependent sRNA silencing is crucial to specify cell fate in the Arabidopsis ovule, and that epigenetic reprogramming in companion cells is necessary for sRNA-dependent silencing in plant gametes.

Embryo and endosperm inherit distinct chromatin and transcriptional states from the female gametes in Arabidopsis.

Whether deposited maternal products are important during early seed development in flowering plants remains controversial. Here, we show that RNA interference-mediated downregulation of transcription is deleterious to endosperm development but does not block zygotic divisions. Furthermore, we show that RNA POLYMERASE II is less active in the embryo than in the endosperm. This dimorphic pattern is established late during female gametogenesis and is inherited by the two products of fertilization. This juxtaposition of distinct transcriptional activities correlates with differential patterns of histone H3 lysine 9 dimethylation, LIKE HETEROCHROMATIN PROTEIN1 localization, and Histone H2B turnover in the egg cell versus the central cell. Thus, distinct epigenetic and transcriptional patterns in the embryo and endosperm are already established in their gametic progenitors. We further demonstrate that the non-CG DNA methyltransferase CHROMOMETHYLASE3 (CMT3) and DEMETER-LIKE DNA glycosylases are required for the correct distribution of H3K9 dimethylation in the egg and central cells, respectively, and that plants defective for CMT3 activity show abnormal embryo development. Our results provide evidence that cell-specific mechanisms lead to the differentiation of epigenetically distinct female gametes in Arabidopsis thaliana. They also suggest that the establishment of a quiescent state in the zygote may play a role in the reprogramming of the young plant embryo.

Reproduction of the biogenic reef-forming honeycomb worm Sabellaria alveolata in Ireland

The stage of development of the gametes of both male and female honeycomb worms Sabellaria alveolata (L.) was examined, using histology, over a 14-month period. Samples came from Howes Strand and Garrettstown, County Cork in south-west Ireland. A clear cycle of both male and female gametogenesis was evident, with a distinct peak in the summer (June–September) when the majority of them were ripe. During October–January a small proportion of males contained abundant spermatozoa, but no mature oocytes were found. Over the whole sampling period the ratio of males:females was 1.4:1. Further south in Roches de la Fosse in France at the centre of its range, two spawnings and an even sex-ratio have been reported.

BiP-mediated polar nuclei fusion is essential for the regulation of endosperm nuclei proliferation in Arabidopsis thaliana

Nuclear fusion is an essential process in the sexual reproduction of animals and plants. In flowering plants, nuclear fusion occurs three times: once during female gametogenesis, when the two polar nuclei fuse to produce the diploid central cell nucleus, and twice during double fertilization. The yeast Ig binding protein (BiP) is a molecular chaperone Hsp70 in the endoplasmic reticulum that regulates nuclear membrane fusion during mating. Here we report that in Arabidopsis thaliana, BiP is involved in the fusion of polar nuclei during female gametophyte development. BiP-deficient mature female gametophytes contain two unfused polar nuclei, in spite of their close contact. This indicates a surprising conservation of BiP function in nuclear fusion between plants and yeasts. We also found that endosperm nuclear division becomes aberrant after fertilization of the BiP-deficient female gametophytes with wild-type pollen. This is experimental evidence for the importance of fusion of the polar nuclei in the proliferation of endosperm nuclei.

Sam68: A New STAR in the Male Fertility Firmament

Male infertility accounts for approximately 50% of the cases of sterile human couples, and in many instances the genetic or molecular defects involved remain unknown. Studies conducted in animal models have elucidated the key role played by RNA-binding proteins and by the posttranscriptional regulation of gene expression during spermatogenesis. Ablation of proteins involved in each of the steps required for the processing and the utilization of messenger RNAs impairs the production of fertile spermatozoa. Recent evidence indicates that the RNA-binding protein Sam68 is absolutely required for the correct progression of spermatogenesis and for male fertility in the mouse. Sam68 belongs to the evolutionary conserved signal transduction and activation of RNA (STAR) family of RNA-binding proteins. The members of this family have been demonstrated to play crucial roles in cell differentiation and development, including male and female gametogenesis. In this review we will summarize the observations gathered on the functions of STAR proteins in different organisms, with particular emphasis on the role of Sam68 in male fertility.

Introductory Overview to the Proceedings of the XXth North American Testis Workshop

Introductory Overview to the Proceedings of the XXth North American Testis Workshop

The MIDASIN and NOTCHLESS genes are essential for female gametophyte development in Arabidopsis thaliana

Female gametophyte development in Arabidopsis thaliana follows a well-defined program that involves many fundamental cellular processes. In this study, we report the involvement of the Arabidopsis thaliana MIDASIN1 (AtMDN1) gene during female gametogenesis through the phenotypic characterization of plants heterozygous for an insertional mdn1 mutant allele. The MDN1 yeast ortholog has previously been shown to encode a non-ribosomal protein involved in the maturation and assembly of the 60S ribosomal subunit. Heterozygous MDN1/mdn1 plants were semisterile and mdn1 allele transmission through the female gametophyte was severely affected. Development of mdn1 female gametophyte was considerably delayed compared to their wild-type siblings. However, delayed mdn1 female gametophytes were able to reach maturity and a delayed pollination experiment showed that a small proportion of the female gametophytes were functional. We also report that the Arabidopsis NOTCHLESS (AtNLE) gene is also required for female gametogenesis. The NLE protein has been previously shown to interact with MDN1 and to be also involved in 60S subunit biogenesis. The introduction of an AtNLE-RNA interference construct in Arabidopsis led to semisterility defects. Defective female gametophytes were mostly arrested at the one-nucleate (FG1) developmental stage. These data suggest that the activity of both AtMDN1 and AtNLE is essential for female gametogenesis progression.

Meiotically asynapsis-induced aneuploidy in autopolyploid Arabidopsis thaliana

The patterns of homologue segregation are the basis for euploidy or aneuploidy formation in diploids and allo-/auto-polyploids. Homologue segregation in diploids resembles that in allopolyploids during meiosis; however, meiotic chromosome behavior in autopolyploids is complicated by multiplication of homologous chromosome components. Obviously, loss of single chromosomes (or segmented chromosomes) frequently leads to abortion of reproductive gametes in diploids and allopolyploids. In contrast, the consequence of chromosome loss in autopolyploids is effortlessly compensated for by the presence of multiplied chromosome complements. Here, we use the meiotically asynaptic gene asy1, in combination with polyploidization, to elucidate aneuploidy formation in autotetraploid Arabidopsis. The results indicate that, due to homologous asynapsis in meiotic prophase I, retarded chromosome losses could induce aneuploidy during gametogenesis in autotetraploid asy1. The severe loss of individual chromosomes probably reaches the haploid genome among selfed or backcrossed progeny, leading to stochastic chromosome loss in Arabidopsis. Reciprocal crosses of autotetraploid asy1 with wild-type prove a pathway of duoparental transmission of aneuploidy (hypoploidy and hyperploidy). Viable hypoploids over-transmit via male gametes; conversely, viable hyperploids transmit mainly in female gametogenesis. This result suggests a more stringent maternal restriction of ploidy transmission in autopolyploid Arabidopsis.

SLOW WALKER2, a NOC1/MAK21 Homologue, Is Essential for Coordinated Cell Cycle Progression during Female Gametophyte Development in Arabidopsis

Morphogenesis requires the coordination of cell growth, division, and cell differentiation. Female gametogenesis in flowering plants, where a single haploid spore undergoes continuous growth and nuclear division without cytokinesis to form an eight-nucleate coenocytic embryo sac before cellularization, provides a good system to study the genetic control of such processes in multicellular organisms. Here, we report the characterization of an Arabidopsis (Arabidopsis thaliana) female gametophyte mutant, slow walker2 (swa2), in which the progression of the mitotic cycles and the synchrony of female gametophyte development were impaired, causing an arrest of female gametophytes at the two-, four-, or eight-nucleate stage. Delayed pollination test showed that a portion of the mutant ovules were able to develop into functional embryo sacs and could be fertilized. SWA2 encodes a nucleolar protein homologous to yeast NUCLEOLAR COMPLEX ASSOCIATED PROTEIN1 (NOC1)/MAINTENANCE OF KILLER21 that, together with NOC2, is involved in preribosome export from the nucleus to the cytoplasm. Similarly, SWA2 can physically interact with a putative Arabidopsis NOC2 homologue. SWA2 is expressed ubiquitously throughout the plant, at high levels in actively dividing tissues and gametophytes. Therefore, we conclude that SWA2 most likely plays a role in ribosome biogenesis that is essential for the coordinated mitotic progression of the female gametophyte.