Behavior of filamentous temperature-sensitive Z2 (FtsZ2) in the male gametophyte during sexual reproduction processes of flowering plants.

Filamentous temperature-sensitive Z (FtsZ) is a critical division protein in bacteria that functions in forming a Z-ring structure to constrict the cell. Since the establishment of the plastid by endosymbiosis of a cyanobacterium into a eukaryotic cell, division via Z-ring formation has been conserved in the plastids of flowering plants. The FtsZ gene was transferred from the cyanobacterial ancestor of plastids to the eukaryotic nuclear genome during evolution, and flowering plants evolved two FtsZ homologs, FtsZ1 and FtsZ2, which are involved in chloroplast division through distinct molecular functions. Regarding the behaviors of FtsZ in nonphotosynthetic cells, the plastid localization of FtsZ1 proteins in the cytoplasm of microspores and pollen vegetative cells but not in generative cells or sperm cells has been reported. On the other hand, the significant accumulation of FtsZ2 transcripts in generative cells has been reported. However, the synthesis of FtsZ2 in the male gamete has not been investigated. Additionally, FtsZ2 behavior has not been analyzed in pollen, a nonphotosynthetic male tissue. Here, we report FtsZ2 protein behaviors in the male gamete by analyzing the localization patterns of GFP-fused protein at various pollen developmental stages and in gametes during the fertilization process. Our results showed that FtsZ2 localization coincided with that of plastids. FtsZ2 protein in male gametes was almost absent, despite the presence of the transcripts. Moreover, transmission of paternal FtsZ2 transcripts to the zygote and endosperm was not observed.

The male gamete membrane protein DMP9/DAU2 is required for double fertilization in flowering plants.

All flowering plants exhibit a unique type of sexual reproduction called 'double fertilization' in which each pollen tube-delivered sperm cell fuses with an egg and a central cell. Proteins that localize to the plasma membrane of gametes regulate one-to-one gamete pairing and fusion between male and female gametes for successful double fertilization. Here, we have identified a membrane protein from Lilium longiflorum generative cells using proteomic analysis and have found that the protein is an ortholog of Arabidopsis DUF679 DOMAIN MEMBRANE PROTEIN 9 (DMP9)/DUO1-ACTIVATED UNKNOWN 2 (DAU2). The flowering plant DMP9 proteins analyzed in this study were predicted to have four transmembrane domains and be specifically expressed in both generative and sperm cells. Knockdown of DMP9 resulted in aborted seeds due to single fertilization of the central cell. Detailed imaging of DMP9-knockdown sperm cells during in vivo and semi-in vitro double fertilization revealed that DMP9 is involved in gamete interaction that leads to correct double fertilization.

Molecular evolutionary analysis of a gender-limited MID ortholog from the homothallic species Volvox africanus with male and monoecious spheroids.

Volvox is a very interesting oogamous organism that exhibits various types of sexuality and/or sexual spheroids depending upon species or strains. However, molecular bases of such sexual reproduction characteristics have not been studied in this genus. In the model species V. carteri, an ortholog of the minus mating type-determining or minus dominance gene (MID) of isogamous Chlamydomonas reinhardtii is male-specific and determines the sperm formation. Male and female genders are genetically determined (heterothallism) in V. carteri, whereas in several other species of Volvox both male and female gametes (sperm and eggs) are formed within the same clonal culture (homothallism). To resolve the molecular basis of the evolution of Volvox species with monoecious spheroids, we here describe a MID ortholog in the homothallic species V. africanus that produces both monoecious and male spheroids within a single clonal culture. Comparison of synonymous and nonsynonymous nucleotide substitutions in MID genes between V. africanus and heterothallic volvocacean species suggests that the MID gene of V. africanus evolved under the same degree of functional constraint as those of the heterothallic species. Based on semi quantitative reverse transcription polymerase chain reaction analyses using the asexual, male and monoecious spheroids isolated from a sexually induced V. africanus culture, the MID mRNA level was significantly upregulated in the male spheroids, but suppressed in the monoecious spheroids. These results suggest that the monoecious spheroid-specific down regulation of gene expression of the MID homolog correlates with the formation of both eggs and sperm in the same spheroid in V. africanus.

GENERATIVE CELL SPECIFIC 1 is essential for angiosperm fertilization.

The double fertilization process in angiosperms is based on the delivery of a pair of sperm cells by the pollen tube (the male gametophyte), which elongates towards an embryo sac (the female gametophyte) enclosing an egg and a central cell. Several studies have described the mechanisms of gametophyte interaction, and also the fertilization process - from pollination to pollen tube acceptance. However, the mechanisms of gamete interaction are not fully understood. Cytological studies have shown that male gametes possess distinct cell-surface structures and genes specific to male gametes have been detected in cDNA libraries. Thus, studies of isolated gametes may offer clues to understanding the sperm-egg interaction. In this study, we identified a novel protein, designated GCS1 (GENERATIVE CELL SPECIFIC 1), using generative cells isolated from Lilium longiflorum pollen. GCS1 possesses a carboxy-terminal transmembrane domain, and homologues are present in various species, including non-angiosperms. Immunological assays indicate that GCS1 is accumulated during late gametogenesis and is localized on the plasma membrane of generative cells. In addition, Arabidopsis thaliana GCS1 mutant gametes fail to fuse, resulting in male sterility and suggesting that GCS1 is a critical fertilization factor in angiosperms.

Laparoscopic lymph node dissection after endoscopic submucosal dissection: a novel and minimally invasive approach to treating early-stage gastric cancer.

BACKGROUND AND OBJECTIVE: Endoscopic submucosal dissection (ESD), a newly developed endoscopic mucosal resection (EMR) technique, can completely cure a differentiated mucosal gastric cancer smaller than 2 cm. For early-stage gastric cancers (EGCs) deviating from the above-mentioned criterion, gastrectomy with lymph node dissection is performed for potential risk of lymph node metastasis (LNM). However, many of surgical EGC cases actually do not have LNM, indicating this surgery may not be necessary for many cases of EGC. To avoid this unnecessary surgery, we have introduced laparoscopic lymph node dissection (LLND) after ESD. Standard gastrectomy with extended lymph node dissection is indicated for patients if LLND reveals LNM. We present our novel approach and the preliminary results of EGC patients having potential risk of LNM. METHODS: Five patients with EGC deviating from the EMR criterion underwent the combination of ESD and LLND. ESD was performed using a newly developed insulation-tipped diathermic knife. Lymph nodes, which were determined on the basis of the location of the primary tumor and lymphatic drainage of the stomach, were removed laparoscopically. The lymphatic drainage was visualized by submucosally injecting indocyanine green (ICG) around the post-ESD ulcerative scars during intraoperative gastroscopy. RESULTS: The ESD enabled en bloc resection without any complications. The resected margins of all the lesions were free of cancer cells vertically and horizontally. LLND was successfully performed without any complications. The mean number of the dissected lymph nodes was 15 (range 6 to 22). In 4 of the 5 patients, the dissected lymph nodes were free of cancer cells, and therefore, the combination of ESD and LLND was considered a definitive treatment. The remaining patient was found to have LNM but chose not to undergo any surgery. During follow-ups, the patients' previous quality of life was restored without any tumor recurrence. CONCLUSIONS: The combination of ESD and LLND enables the complete resection of the primary tumor and the histologic determination of lymph node status. This combination treatment is a potential, minimally invasive method, and may obviate unnecessary gastrectomy without compromising curability for EGC patients having the potential risk of LNM.

Identification of higher plant GlsA, a putative morphogenesis factor of gametic cells.

GlsA has been identified in an asexual-reproductive-cell (gonidia)-deficient mutant of Volvox as a chaperone-like protein essential for gonidia production. In this study, we isolated an angiosperm glsA (LlglsA) gene expressed during Lilium longiflorum pollen development. Immunoblot analyses showed that the strong LlGlsA expression occurred in the generative cell and its pattern during pollen development corresponded to that of alpha-tubulin. Morphological analyses succeeded in visualizing the dispersion of the strong LlGlsA signal in developing generative cells. In addition, multiple-immunofluorescence staining of LlGlsA and alpha-tubulin revealed that some of the dot-like LlGlsA signals were co-localized with microtubule filaments. From those results, we suggest that angiosperm GlsA functions as a chaperone modifying various structures during male gametic cell formation.