SATB1 establishes ameloblast cell polarity and regulates directional amelogenin secretion for enamel formation

Background Polarity is necessary for epithelial cells to perform distinct functions at their apical and basal surfaces. Oral epithelial cell-derived ameloblasts at secretory stage (SABs) synthesize large amounts of enamel matrix proteins (EMPs), largely amelogenins. EMPs are unidirectionally secreted into the enamel space through their apical cytoplasmic protrusions, or Tomes’ processes (TPs), to guide the enamel formation. Little is known about the transcriptional regulation underlying the establishment of cell polarity and unidirectional secretion of SABs. Results The higher-order chromatin architecture of eukaryotic genome plays important roles in cell- and stage-specific transcriptional programming. A genome organizer, special AT-rich sequence-binding protein 1 (SATB1), was discovered to be significantly upregulated in ameloblasts compared to oral epithelial cells using a whole-transcript microarray analysis. The Satb1−/− mice possessed deformed ameloblasts and a thin layer of hypomineralized and non-prismatic enamel. Remarkably, Satb1−/− ameloblasts at the secretory stage lost many morphological characteristics found at the apical surface of wild-type (wt) SABs, including the loss of Tomes’ processes, defective inter-ameloblastic adhesion, and filamentous actin architecture. As expected, the secretory function of Satb1−/− SABs was compromised as amelogenins were largely retained in cells. We found the expression of epidermal growth factor receptor pathway substrate 8 (Eps8), a known regulator for actin filament assembly and small intestinal epithelial cytoplasmic protrusion formation, to be SATB1 dependent. In contrast to wt SABs, EPS8 could not be detected at the apical surface of Satb1−/− SABs. Eps8 expression was greatly reduced in small intestinal epithelial cells in Satb1−/− mice as well, displaying defective intestinal microvilli. Conclusions Our data show that SATB1 is essential for establishing secretory ameloblast cell polarity and for EMP secretion. In line with the deformed apical architecture, amelogenin transport to the apical secretory front and secretion into enamel space were impeded in Satb1−/− SABs resulting in a massive cytoplasmic accumulation of amelogenins and a thin layer of hypomineralized enamel. Our studies strongly suggest that SATB1-dependent Eps8 expression plays a critical role in cytoplasmic protrusion formation in both SABs and in small intestines. This study demonstrates the role of SATB1 in the regulation of amelogenesis and the potential application of SATB1 in ameloblast/enamel regeneration.

Mitochondria-enriched protrusions are associated with brain and intestinal stem cells in Drosophila

Brain stem cells stop dividing in late Drosophila embryos and begin dividing again in early larvae after feeding induces reactivation. Quiescent neural stem cells (qNSCs) display an unusual cytoplasmic protrusion that is no longer present in reactivated NSCs. The protrusions join the qNSCs to the neuropil, brain regions that are thought to maintain NSCs in an undifferentiated state, but the function of the protrusions is not known. Here we show that qNSC protrusions contain clustered mitochondria that are likely maintained in position by slow forward-and-backward microtubule growth. Larvae treated with a microtubule-stabilizing drug show bundled microtubules and enhanced mitochondrial clustering in NSCs, together with reduced qNSC reactivation. We further show that intestinal stem cells contain mitochondria-enriched protrusions. The qNSC and intestinal stem-cell protrusions differ from previously reported cytoplasmic extensions by forming stem-cell-to-niche mitochondrial bridges that could potentially both silence genes and sense signals from the stem cell niche.

Soluble sperm extract specifically recapitulates the initial phase of the Ca2+ response in the fertilized oocyte of P. occelata following a G-protein/ PLCβ signaling pathway

SummaryMatured oocytes of the annelidan worm Pseudopotamilla occelata are fertilized at the first metaphase of the meiotic division. During the activation by fertilizing spermatozoa, the mature oocyte shows a two-step intracellular Ca2+ increase. Whereas the first Ca2+ increase is localized and appears to utilize the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores, the second Ca2+ increase is global and involves Ca2+ influx via voltage-gated Ca2+ channels on the entire surface of the oocyte. To study how sperm trigger the Ca2+ increases during fertilization, we prepared soluble sperm extract (SE) and examined its ability to induce Ca2+ increases in the oocyte. The SE could evoke a Ca2+ increase in the oocyte when it was added to the medium, but not when it was delivered by microinjection. However, the second-step Ca2+ increase leading to the resumption of meiosis did not follow in these eggs. Local application of SE induced a non-propagating Ca2+ increase and formed a cytoplasmic protrusion that was similar to that created by the fertilizing sperm at the first stage of the Ca2+ response, important for sperm incorporation into the oocyte. Our results suggest that the fertilizing spermatozoon may trigger the first-step Ca2+ increase before it fuses with the oocyte in a pathway that involves the G-protein-coupled receptor and phospholipase C. Thus, the first phase of the Ca2+ response in the fertilized egg of this species is independent of the second phase of the Ca2+ increase for egg activation.

42 ASSISTED ENUCLEATION IN GOLDEN HAMSTER OOCYTES TREATED WITH COLCHICINE AND DEMECOLCINE

The golden hamster is an excellent experimental animal in many research fields. In an effort to establish experimental protocols necessary for cloning golden hamsters, optimized conditions for induced cytoplasmic protrusion and assisted enucleation of golden hamster oocytes with different concentrations of colchicine and demecolcine were examined in this study. The Golden hamsters (female, six weeks of age) were superovulated with eCG (30 IU, ip) followed by hCG (30 IU, ip) at intervals of 72 h. Hamsters were sacrificed at 13.5 h, 15 h and 18 h after hCG injection. The different ages of cumulus–oocyte complexes (COCs) were collected from oviducts and cumulus cells were removed with 0.1% hyaluronidase. (1) denuded oocytes of different ages were treated with 2.5 μg mL–1, 5 μg mL–1, and 10 μg mL–1 of colchicine for 4 h in M199TE, and they were examined each hour; (2) denuded oocytes of different ages were treated with 0.02 μg mL–1, 0.04 μg mL–1, 0.06 μg mL–1, 0.1 μg mL–1, 0.2 μg mL, 0.4 μg mL–1, and 0.6 μg mL–1 of demecolcine for 1 h in M199TE, and they were examined every 15 min; 3) according to the results of processes (1) and (2), cytoplasmic protrusions of oocytes treated with 10 μg mL–1 of colchicine (60 oocytes) or 0.4 μg mL–1 demecolcine (88 oocytes) for 1 h were removed with a micromanipulation pipette. Then the oocytes were examined by being stained with Hoechst 33342, and the percentage of assisted enucleation was compared with that of blind enucleation in which position of the chromosomes was indirectly determined by the location of the first polar body. The results showed that: (1) about 90% of oocytes at 13.5 h and 15 h post hCG injection were induced to form protrusions under the treatment of 10 μg mL–1 of colchicine for 1 h; (2) oocytes of 13.5 h post hCG were very sensive to demecolcine, even the treatment of 0.02 μg mL–1 for 1 h could also induce cytoplasmic protrusions (45.38% of 31 oocytes); (3) when treated with 0.4 μg mL–1 of demecolcine for 1 h, the cytoplasmic protrusion rate of oocytes of 13.5 h–18 h post-hCG could reach to 99–100%; (4) when the cytoplasmic protrusions induced by 10 μg mL–1 of colchicine or 0.4 μg mL–1 of demecolcine for 1 h were removed, assisted enucleation rates were over 80%, significantly higher (P < 0.05) than blind enucleation (31.53% of 60 oocytes). In conclusion, the results of this study demonstrate that both colchicine and demecolcine can induce the hamster oocytes to form cytoplasmic protrusions. The treatments of 10 μg mL–1 of colchicine or 0.4 μg mL–1 of demecolcine for 1 h were the best conditions to induce oocytes (13.5 h–18 h post hCG) to form protrusion. Also, the assisted enucleation rate with colchicine and demecolcine is much higher than that of blind enucleation. These results define conditions for chemical assisted enucleation and should facilitate the development of cloned golden hamsters as an animal model for human diseases.

Ultrastructure of spermiogenesis and mature spermatozoon of Skrjabinoporus merops (Cyclophyllidea, Metadilepididae)

The ultrastructure of the mature spermatozoon and the spermiogenesis of a cestode belonging to the family Metadilepididae is described for the first time. The mature spermatozoon of Skrjabinoporus merops is characterized by twisted peripheral microtubules, the presence of a single crested body, periaxonemal sheath and electron-dense rods, and the absence of intracytoplasmic walls and inclusions (glycogen or proteinaceous granules); no peripheral microtubules where nucleus contacts the external plasma membrane. Four morphologically distinct regions of the mature spermatozoon are differentiated. The proximal part (Region I) contains a single crested body, periaxonemal sheath is absent in some (proximal) sections and is present in others situated closer to the nucleus. The central Region II is nucleated, and is followed by Region III that contains a periaxonemal sheath. The distal pole, Region IV, is characterized by disintegration of the axoneme. Spermiogenesis follows the type III pattern (Bâ and Marchand 1995) although in S. merops a slight flagellar rotation is observed. The differentiation zone is characterized by the absence of striated roots and intercentriolar body; two centrioles are present, one of which gives rise to a free flagellum. The latter rotates and undergoes proximodistal fusion with the cytoplasmic protrusion of the differentiation zone. Spermiological characters of S. merops are similar to those of the families Taeniidae and Catenotaeniidae. The mature spermatozoon differs from those of the Dilepididae (where the metadilepidid species have previously been classified) by the lack of glycogen.

Ultrastructure of spermiogenesis and mature spermatozoon of Angularella beema (Clerc, 1906) (Cestoda, Cyclophyllidea, Dilepididae)

The ultrastructure of the spermiogenesis of a dilepidid cestode species is described for the first time. The spermiogenesis of Angularella beema is characterised by absence of both flagellar rotation and proximodistal fusion. The differentiation zone is surrounded by cortical microtubules and is delimited by a ring of arching membranes. It contains two centrioles, one of which develops the axoneme that grows directly into the elongating cytoplasmic protrusion. This pattern of spermiogenesis was described as the Type IV spermiogenesis of cestodes. Among cestodes, similar pattern of spermiogenesis is known in the family Hymenolepididae and in some representatives of the family Anoplocephalidae. The mature spermatozoon of A. beema consists of five regions differing in their ultrastructural characteristics. It is characterised by the presence of cortical microtubules (spirally arranged at angle of 30–40° to the spermatozoon axis) and a single crested body. There is a periaxonemal sheath in certain parts of the spermatozoon as well as glycogen-like granules between the periaxonemal sheath and the cortical microtubules. The comparisons of the mature spermatozoon of A. beema with those of other two dilepidid species (Dilepis undula and Molluscotaenia crassiscolex) demonstrate some variation within the family: presence of periaxonemal sheath in A. beema and D. undula and its absence in M. crassiscolex; presence of electron-dense rods in D. undula and their absence in A. beema.

Sperm-oocyte interaction in the sea-urchin

A study was made of the electrical and morphological changes in the sea-urchin oocyte during interaction with spermatozoa. The first event, a small step depolarization accompanied by a 20–40% decay in input resistance occurs within seconds of attachment. The evidence suggests that this electrical event is the result of sperm-oocyte fusion, and that the ion channels that lower the resistance across the oocyte-sperm complex are located in the sperm plasma membrane. This primary electrical event does not necessarily lead to sperm incorporation. A second, determinative, event occurs at 50 s, which leads to sperm entry and the formation of a cytoplasmic protrusion at the site of sperm entry. This second event probably results from the transfer of a soluble component from the spermatozoon into the oocyte cytoplasm, which leads to sperm incorporation and formation of the protrusion. The changes in the oocyte following insemination are compared with the events of egg activation.

Aspects of silicification in wall morphogenesis of diatoms

The siliceous cell wall of diatoms is formed in a silica deposition vesicle that is delimited by a membrane, the silicalemma. Once the siliceous wall matures, it is expelled and a new plasmalemma is formed underneath. Underlying wall formation is a multitude of events and processes, some of which are now known. A comparative study on wall morphogenesis in seven centric diatoms leads to the following conclusions: (1) the silica deposition vesicle is formed by the coalescence of small vesicles; (2) the silicalemma becomes part of the organic casing of the mature siliceous wall; (3) at least four morphological forms of deposited silica can be seen during the development of wall components; (4) microtubules, serving as cytoskeletons, are associated with the formation of certain wall components initiated from a cytoplasmic protrusion. These events are discussed in detail.

Circus movements and blebbing locomotion in dissociated embryonic cells of an amphibian, Xenopus laevis

Circus movements, which involve the circumferential rotation of a hyaline cytoplasmic protrusion, occur in cells obtained by EDTA dissociation of gastrula-stage Xenopus laevis embryos. Only a few dissociated blastula-stage cells show circus movements, more early gastrula-stage cells show them, and nearly all late gastrula-stage cells show them. Circus movements cease in cells prior to mitosis and begin again in daughter cells after mitosis is completed. In early gastrulae, only 17% of prospective endodermal cells show circus movements while 79% of prospective mesodern, archenteric roof, and posterior neural ectoderm do so. Isolated cells as well as groups of cells in vitro are often propelled by circus movements. There is an obvious antagonism between cell contact and circus movements. The morphogenetic significance of circus movements and blebbing locomotion is discussed.