0000000000040408
AUTHOR
Bärbel Diehl-seifert
Silica as a morphogenetically active inorganic polymer.
At present the scaffolds used for bioprinting of cells do not elicit morphogenetic responses in the cells. In the present study we approached a solution by studying the effect of an inorganic silica supplement added to an Na-alginate matrix. Bone- and osteoblast-like SaOS-2 cells were embedded into this organic polymeric matrix which was additionally enriched with 400 μM prehydrolyzed TEOS [tetra-ethoxy-silane], a source of ortho-silicate. In this silica-based matrix the cells synthesized hydroxyapatite crystallites after exposure to a mineralization activation cocktail composed of β-glycerophosphate, ascorbic acid and dexamethasone. The degree of hydroxyapatite synthesis, determined by sta…
Mineralization of bone-related SaOS-2 cells under physiological hypoxic conditions
Inorganic polyphosphate (polyP) is a physiological energy-rich polymer with multiple phosphoric anhydride bonds. In cells such as bone-forming osteoblasts, glycolysis is the main pathway generating metabolic energy in the form of ATP. In the present study, we show that, under hypoxic culture conditions, the growth/viability of osteoblast-like SaOS-2 cells is not impaired. The addition of polyP to those cells, administered as amorphous calcium polyP nanoparticles (aCa-polyP-NP; approximate size 100 nm), significantly increased the proliferation of the cells. In the presence of polyP, the cells produce significant levels of lactate, the end product of anaerobic glycolysis. Under those conditi…
Identification and purification of a stress associated nuclear carbohydrate binding protein (Mr 33000) from rat liver by application of a new photoreactive carbohydrate probe
A photoreactive alpha-D-glucose probe has been designed for the specific detection of carbohydrate binding proteins (CBPs). The probe consists of four parts: (i) an alpha-D-glucose moiety; (ii) the digoxigenin tag; (iii) the photoreactive cross-linker; and (iv) the lysyl-lysine backbone. After incubation with lectins in the dark, the probe is activated and cross-linked to the CBPs after being treated by several flashes. Using this method we have identified a new alpha-D-glucose CBP of M(r) = 33,000, termed CBP33, in the nuclei of rats exposed to transient immobilization stress. Monoclonal antibodies were raised against the partially purified protein and subsequently used to enrich CBP33. It…
A galectin links the aggregation factor to cells in the sponge (Geodia cydonium) system.
The cDNA for the full-length lectin from the marine sponge Geodia cydonium was cloned. Analysis of the deduced aa sequence revealed that this lectin belongs to the group of galectins. The full-length galectin, which was obtained also in a recombinant form, has an M(r) of 20,877; in the processed form it is a 15 kDa polypeptide. The enriched aggregation factor from G.cydonium also was determined to contain, besides minimal amounts of the galectin, a 140 kDa polypeptide which is involved in cell-cell adhesion. Monoclonal antibodies have been raised against this protein; Fab' fragments prepared from them abolished cell-cell reaggregation. Cell reaggregation experiments revealed that the aggreg…
Nonenzymatic Transformation of Amorphous CaCO3 into Calcium Phosphate Mineral after Exposure to Sodium Phosphate in Vitro: Implications for in Vivo Hydroxyapatite Bone Formation.
Studies indicate that mammalian bone formation is initiated at calcium carbonate bioseeds, a process that is driven enzymatically by carbonic anhydrase (CA). We show that amorphous calcium carbonate (ACC) and bicarbonate (HCO3 (-) ) cause induction of expression of the CA in human osteogenic SaOS-2 cells. The mineral deposits formed on the surface of the cells are rich in C, Ca and P. FTIR analysis revealed that ACC, vaterite, and aragonite, after exposure to phosphate, undergo transformation into calcium phosphate. This exchange was not seen for calcite. The changes to ACC, vaterite, and aragonite depended on the concentration of phosphate. The rate of incorporation of phosphate into ACC, …
Retinol encapsulated into amorphous Ca2+ polyphosphate nanospheres acts synergistically in MC3T3-E1 cells
Both the quality and quantity of collagen, the major structural component of the skin, decrease in aging skin. We succeeded to encapsulate retinol into amorphous inorganic polyphosphate (polyP) nanoparticles together with calcium ions ("aCa-polyP-NP"), under formation of amorphous Ca-polyP/retinol nanospheres ("retinol/aCa-polyP-NS"). The globular nanospheres are not cytotoxic, show an almost uniform size of ≈ 45 nm and have a retinol content of around 25%. Both components of those nanospheres, retinol and the aCa-polyP-NP, if administered together, caused a strong increase in proliferation of mouse calvaria MC3T3 cells. The expressions of collagen types I, II and III genes, but not the exp…
A novel tunicate (Botryllus schlosseri) putative C-type lectin features an immunoglobulin domain.
We have cloned a putative C-type lectin of Botryllus schlosseri [Ascidiacea], whose deduced protein of 333 amino acids features three building blocks: (i) a Greek-key motif signature at the amino-terminus, (ii) a C-type lectin domain signature, and (iii) an immunoglobulin (Ig) domain at the carboxyl terminus. This C-type lectin was termed BSCLT. Similarity searches revealed that the Ig domain in BSCLT, which is evidently not polymorphic, is best classified as an Intermediate-type Ig domain. Rabbit antibodies, raised against recombinant BSCLT, cross-reacted in a Western blot with a 38-kD polypeptide in tunicate crude extract. Presumably, this bimodal tunicate protein is the first description…
Potential biological role of laccase from the sponge Suberites domuncula as an antibacterial defense component
Abstract Background Laccases are copper-containing enzymes that catalyze the oxidation of a wide variety of phenolic substrates. Methods We describe the first poriferan laccase from the marine demosponge Suberites domuncula. Results This enzyme comprises three characteristic multicopper oxidase homologous domains. Immunohistological studies revealed that the highest expression of the laccase is in the surface zone of the animals. The expression level of the laccase gene is strongly upregulated after exposure of the animals to the bacterial endotoxin lipopolysaccharide. To allow the binding of the recombinant enzyme to ferromagnetic nanoparticles, a recombinant laccase was prepared which con…
Biosilica-loaded poly(ϵ-caprolactone) nanofibers mats provide a morphogenetically active surface scaffold for the growth and mineralization of the osteoclast-related SaOS-2 cells.
Bioprinting/3D cell printing procedures for the preparation of scaffolds/implants have the potential to revolutionize regenerative medicine. Besides biocompatibility and biodegradability, the hardness of the scaffold material is of critical importance to allow sufficient mechanical protection and, to the same extent, allow migration, cell–cell, and cell–substrate contact formation of the matrix-embedded cells. In the present study, we present a strategy to encase a bioprinted, cell-containing, and soft scaffold with an electrospun mat. The electrospun poly(e-caprolactone) (PCL) nanofibers mats, containing tetraethyl orthosilicate (TEOS), were subsequently incubated with silicatein. Silicate…
Silicate modulates the cross-talk between osteoblasts (SaOS-2) and osteoclasts (RAW 264.7 cells): inhibition of osteoclast growth and differentiation
It has been shown that inorganic monomeric and polymeric silica/silicate, in the presence of the biomineralization cocktail, increases the expression of osteoprotegerin (OPG) in osteogenic SaOS-2 sarcoma cells in vitro. In contrast, silicate does not affect the steady-state gene expression level of the osteoclastogenic ligand receptor activator of NF-κB ligand (RANKL). In turn it can be expected that the concentration ratio of the mediators OPG/RANKL increases in the presence of silicate. In addition, silicate enhances the growth potential of SaOS-2 cells in vitro, while it causes no effect on RAW 264.7 cells within a concentration range of 10-100 µM. Applying a co-cultivation assay system,…
Increased Expression of Integrin and Receptor Tyrosine Kinase Genes During Autograft Fusion in the SpongeGeodia cydonium
Recently cDNAs coding for cell surface molecules have been isolated from sponges. The molecules for alpha-integrin, galectin, and receptor tyrosine kinase (RTK), obtained from the marine sponge, Geodia cydonium, have been described earlier. In the present study also the cDNA for one putative beta-integrin has been identified from G. cydonium. The deduced aa sequence comprises the characteristic signatures, found in other metazoan beta-integrin molecules; the estimated size is 95,215 Da. To obtain first insights into the molecular events which proceed during autograft fusion, the expressions of these genes were determined on transcriptional and translational level. The cDNAs as well as antib…
Isoquercitrin and polyphosphate co-enhance mineralization of human osteoblast-like SaOS-2 cells via separate activation of two RUNX2 cofactors AFT6 and Ets1.
Isoquercitrin, a dietary phytoestrogen, is a potential stimulator of bone mineralization used for prophylaxis of osteoporotic disorders. Here we studied the combined effects of isoquercitrin, a cell membrane permeable 3-O-glucoside of quercetin, and polyphosphate [polyP], a naturally occurring inorganic polymer inducing bone formation, on mineralization of human osteoblast-like SaOS-2 cells. Both compounds isoquercitrin and polyP induce at non-toxic concentrations the mineralization process of SaOS-2 cells. Co-incubation experiments revealed that isoquercitrin (at 0.1 and 0.3μM), if given simultaneously with polyP (as Ca(2+) salt; at 3, 10, 30 and 100μM) amplifies the mineralization-enhanci…
Transport of mRNA from Nucleus to Cytoplasm
Publisher Summary Transport of mRNP (messenger ribonucleoprotein) from nucleus to cytoplasm plays an important role in gene expression in eukaryotic cells. This chapter focuses on energy-(ATP)-dependent mRNP transport. Nucleocytoplasmic transport of ribosomal RNA can also be induced by ATP, but also occurs by varying [Ca 2+ ]:[Mg 2+ ]. Release of ribosomal RNPs seems to be accompanied by an expansion of the nucleus. Nucleocytoplasmic transport of mRNA seems to be also distinct from the export of tRNA or the exchange of snRNPs and proteins across the nuclear envelope. Nucleocytoplasmic transport of tRNA seems to involve a facilitated diffusion mechanism, showing saturability and sequence spe…
Amorphous polyphosphate–hydroxyapatite: A morphogenetically active substrate for bone-related SaOS-2 cells in vitro
There is increasing evidence that inorganic calcium-polyphosphates (polyP) are involved in human bone hydroxyapatite (HA) formation. Here we investigated the morphology of the particles, containing calcium phosphate (CaP) with different concentrations of various Na-polyP concentrations, as well as their effects in cell culture. We used both SaOS-2 cells and human mesenchymal stem cells. The polymeric phosphate readily binds calcium ions under formation of insoluble precipitates. We found that addition of low concentrations of polyP (10wt.%, referred to the CaP deposits) results in an increased size of the HA crystals. Surprisingly, at higher polyP concentrations (10wt.%) the formation of cr…
Cytochalasin B selectively releases ovalbumin mRNA precursors but not the mature ovalbumin mRNA from hen oviduct nuclear matrix
Hen oviduct nuclear matrix-bound mature ovalbumin mRNA is released from the matrix in the presence of ATP, while the ovalbumin mRNA precursors remain bound to this structure. Detachment of the mature mRNA from the matrix by ATP as well as ATP-dependent efflux of mRNA from isolated nuclei were found to be inhibited by cytochalasin B. On the other hand, in the absence of ATP, cytochalasin B exclusively caused the release (and nucleocytoplasmic efflux) of the ovalbumin messenger precursors, but not of the mature mRNA. After cytochalasin B treatment, actin could be detected in the matrix supernatant. Phalloidin which stabilizes actin filaments did not cause RNA liberation in the absence of ATP,…
Sponge Bcl-2 homologous protein (BHP2-GC) confers distinct stress resistance to human HEK-293 cells
It is established that sponges, the phylogenetically oldest still extant phylum of Metazoa, possess key molecules of the apoptotic pathways, that is members from the Bcl-2 family and a pro-apoptotic molecule with death domains. Here we report on transfection studies of human cells with a sponge gene, GCBHP2. Sponge tissue was exposed to heat shock and tributyltin, which caused an upregulation of gene expression of GCBHP2. The cDNA GCBHP2 was introduced into human HEK-293 cells and mouse NIH-3T3 cells; the stable transfection was confirmed by the identification of the transcripts, by Western blotting as well as by immunofluorescence using antibodies raised against the recombinant polypeptide…
Modular Small Diameter Vascular Grafts with Bioactive Functionalities.
We report the fabrication of a novel type of artificial small diameter blood vessels, termed biomimetic tissue-engineered blood vessels (bTEBV), with a modular composition. They are composed of a hydrogel scaffold consisting of two negatively charged natural polymers, alginate and a modified chitosan, N,O-carboxymethyl chitosan (N,O-CMC). Into this biologically inert scaffold two biofunctionally active biopolymers are embedded, inorganic polyphosphate (polyP) and silica, as well as gelatin which exposes the cell recognition signal, Arg-Gly-Asp (RGD). These materials can be hardened by exposure to Ca(2+) through formation of Ca(2+) bridges between the polyanions, alginate, N,O-CMC, and polyP…
Engineering a morphogenetically active hydrogel for bioprinting of bioartificial tissue derived from human osteoblast-like SaOS-2 cells.
Abstract Sodium alginate hydrogel, stabilized with gelatin, is a suitable, biologically inert matrix that can be used for encapsulating and 3D bioprinting of bone-related SaOS-2 cells. However, the cells, embedded in this matrix, remain in a non-proliferating state. Here we show that addition of an overlay onto the bioprinted alginate/gelatine/SaOS-2 cell scaffold, consisting of agarose and the calcium salt of polyphosphate [polyP·Ca 2+ -complex], resulted in a marked increase in cell proliferation . In the presence of 100 μ m polyP·Ca2+ -complex, the cells proliferate with a generation time of approximately 47–55 h. In addition, the hardness of the alginate/gelatin hydrogel substantially i…
Cloning and expression of new receptors belonging to the immunoglobulin superfamily from the marine sponge Geodia cydonium
A cDNA encoding a receptor tyrosine kinase (RTK) was previously cloned and expressed from the marine sponge (Porifera) Geodia cydonium. In addition to the two intracellular regions characteristic for RTKs, two immunoglobulin (Ig)-like domains are found in the extracellular part of the sponge RTK. In the present study it is shown that no further Ig-like domain is present in the upstream region of the cDNA as well as of the gene hitherto known from the sponge RTK. Two different full-length cDNAs have been isolated and characterized in the present study, which possess two Ig-like domains, one transmembrane segment, and only a short intracellular part, without a TK domain. The two deduced polyp…
Dual effect of inorganic polymeric phosphate/polyphosphate on osteoblasts and osteoclasts in vitro
Inorganic polymeric phosphate/polyphosphate (polyP) is a natural polymer existing in both pro- and eukaryotic systems. In the present study the effect of polyP as well as of polyP supplied in a stoichiometric ratio of 2 m polyP:1 m CaCl2 [polyP (Ca2+ complex)] on the osteoblast-like SaOS-2 cells and the osteoclast-like RAW 264.7 cells was determined. Both polymers are non-toxic for these cells up to a concentration of 100 µm. In contrast to polyP, polyP (Ca2+ complex) significantly induced hydroxyapatite formation at a concentration > 10 µm, as documented by alizarin red S staining and scanning electron microscopic (SEM) inspection. Furthermore, polyP (Ca2+ complex) triggered in SaOS-2 cell…
Cloning and expression of the putative aggregation factor from the marine sponge Geodia cydonium.
Sponges (phylum Porifera) have extensively been used as a model system to study cell-cell interaction on molecular level. Recently, we identified and cloned the putative aggregation receptor (AR) of the sponge Geodia cydonium, which interacts in a heterophilic way with the aggregation factor (AF) complex. In the present study, antibodies against this complex have been raised that abolish the adhesion function of the enriched sponge AF, the AF-Fraction 6B. Using this antibody as a tool, a complete 1.7 kb long cDNA, GEOCYAF, could be isolated from a cDNA library that encodes the putative AF. Its deduced aa sequence in the N-terminal section comprises high similarity to amphiphysin/BIN1 sequen…
Effect of bioglass on growth and biomineralization of SaOS-2 cells in hydrogel after 3D cell bioprinting.
We investigated the effect of bioglass (bioactive glass) on growth and mineralization of bone-related SaOS-2 cells, encapsulated into a printable and biodegradable alginate/gelatine hydrogel. The hydrogel was supplemented either with polyphosphate (polyP), administered as polyP • Ca2+-complex, or silica, or as biosilica that had been enzymatically prepared from ortho-silicate by silicatein. These hydrogels, together with SaOS-2 cells, were bioprinted to computer-designed scaffolds. The results revealed that bioglass (nano)particles, with a size of 55 nm and a molar ratio of SiO2 : CaO : P2O5 of 55 : 40 : 5, did not affect the growth of the encapsulated cells. If silica, biosilica, or polyP …
Polyphosphate as a Bioactive and Biodegradable Implant Material: Induction of Bone Regeneration in Rats
Inorganic polyphosphate (polyP) is a naturally occurring polymer that is bioresorbable and anabolically active on bone forming cells in vitro. In order to demonstrate if polyP also shows morphogenetic activity in vivo, animal studies are performed applying the rat calvarial defect model. Poly(D,L-lactide-co-glycolide) (PLGA) microspheres with a narrow size distribution (≈820 μm) are prepared, containing either encapsulated polyP or β-tricalcium phosphate (β-TCP), used as a reference material. Discs are prepared from the microspheres and inserted into 10 mm large defects created in the calvaria of rats. Both the formation of COL-I and the expression of ALP is upregulated, as well as the exte…
Biologizing titanium alloy implant material with morphogenetically active polyphosphate
As a further step towards a new generation of bone implant materials, we developed a procedure for biological functionalization of titanium alloy surfaces with inorganic calcium polyphosphate (Ca-polyP). This polymer has been demonstrated to exhibit morphogenetic activity. The coating of titanium oxidized Ti-6Al-4V scaffolds with biologically active amorphous Ca-polyP microparticles is formed by Ca2+ ion bridges to the silane coupling agent APTMS. This surface is durable and stable as an almost homogeneous Ca-polyP layer onto the metal. The homogenously coated Ca-polyP titanium scaffold was found to be biologically active and supported the growth and functional activity of bone cell-related…
Electrospun bioactive mats enriched with Ca-polyphosphate/retinol nanospheres as potential wound dressing
Background While electrospun materials have been frequently used in tissue engineering no wound dressings exist that significantly improved wound healing effectively. Methods We succeeded to fabricate three-dimensional (3D) electrospun poly(D,l-lactide) (PLA) fiber mats into which nanospheres, formed from amorphous calcium polyphosphate (polyP) nanoparticles (NP) and encapsulated retinol (“retinol/aCa-polyP-NS” nanospheres [NS]), had been incorporated. Results Experiments with MC3T3-E1 cells revealed that co-incubation of the cells with Ca-polyP together with retinol (or incubation with retinol/aCa-polyP-NS) resulted in a significant synergistic effect on cell growth compared with particle-…
Innate Immune Defense of the Sponge Suberites domuncula against Bacteria Involves a MyD88-dependent Signaling Pathway
Sponges (phylum Porifera) are the phylogenetically oldest metazoa; as filter feeders, they are abundantly exposed to marine microorganisms. Here we present data indicating that the demosponge Suberites domuncula is provided with a recognition system for Gram-negative bacteria. The lipopolysaccharide (LPS)-interacting protein was identified as a receptor on the sponge cell surface, which recognizes the bacterial endotoxin LPS. The cDNA was isolated, and the protein (Mr 49,937) was expressed. During binding to LPS, the protein dimerizes and interacts with MyD88, which was also identified and cloned. The sponge MyD88 (Mr 28,441) is composed of two protein interaction domains, a Toll/interleuki…
The Marine Sponge-Derived Inorganic Polymers, Biosilica and Polyphosphate, as Morphogenetically Active Matrices/Scaffolds for the Differentiation of Human Multipotent Stromal Cells: Potential Application in 3D Printing and Distraction Osteogenesis
The two marine inorganic polymers, biosilica (BS), enzymatically synthesized from ortho-silicate, and polyphosphate (polyP), a likewise enzymatically synthesized polymer consisting of 10 to >100 phosphate residues linked by high-energy phosphoanhydride bonds, have previously been shown to display a morphogenetic effect on osteoblasts. In the present study, the effect of these polymers on the differential differentiation of human multipotent stromal cells (hMSC), mesenchymal stem cells, that had been encapsulated into beads of the biocompatible plant polymer alginate, was studied. The differentiation of the hMSCs in the alginate beads was directed either to the osteogenic cell lineage by …
Emergence and Disappearance of an Immune Molecule, an Antimicrobial Lectin, in Basal Metazoa
Sponges (phylum Porifera) represent the evolutionarily oldest metazoans that comprise already a complex immune system and are related to the crown taxa of the protostomians and the deuterostomians. Here, we demonstrate the existence of a tachylectin-related protein in the demosponge Suberites domuncula, termed Suberites lectin. The MAPK pathway was activated in response to lipopolysaccharide treatment of the three-dimensional cell aggregates, the primmorphs; this process was abolished by the monosaccharide D-GlcNAc. The cDNA encoding the S. domuncula lectin was identified and cloned; it comprises 238 amino acids (26 kDa) in the open reading frame. The deduced protein has one potential trans…
Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Ca2+ level in osteoblasts (SaOS-2 cells) in vitro
Inorganic polymeric phosphate is a physiological polymer that accumulates in bone cells. In the present study osteoblast-like SaOS-2 cells were exposed to this polymer, complexed in a 2:1 stoichiometric ratio with Ca(2+), polyP (Ca(2+) salt). At a concentration of 100 μM, polyP (Ca(2+) salt) caused a strong increase in the activity of the alkaline phosphatase and also an induction of the steady-state expression of the gene encoding this enzyme. Comparative experiments showed that polyP (Ca(2+) salt) can efficiently replace β-glycerophosphate in the in vitro hydroxyapatite (HA) biomineralization assay. In the presence of polyP (Ca(2+) salt) the cells extensively form HA crystallites, which r…
The putative sponge aggregation receptor. Isolation and characterization of a molecule composed of scavenger receptor cysteine-rich domains and short consensus repeats.
Porifera (sponges) are the oldest extant metazoan phylum. Dissociated sponge cells serve as a classic system to study processes of cell reaggregation. The reaggregation of dissociated cells is mediated by an extracellularly localized aggregation factor (AF), based on heterophilic interactions of the third order; the AF bridges two cells by ligating a cell-surface-bound aggregation receptor (AR). In the present study we report cloning, expression and immunohistochemical localization of a polypeptide from the marine sponge Geodia cydonium, which very likely represents the AR. The presumed AR gene gives rise to at least three forms of alternatively spliced transcripts of 6.5, 4.9 and 3.9 kb, a…
Electrospinning of Bioactive Wound-Healing Nets
The availability of appropriate dressings for treatment of wounds, in particular chronic wounds, is a task that still awaits better solutions than provided by currently applied materials. The method of electrospinning enables the fabrication of novel materials for wound dressings due to the high surface area and porosity of the electrospun meshes and the possibility to include bioactive ingredients. Recent results show that the incorporation of biologically active inorganic polyphosphate microparticles and microspheres and synergistically acting retinoids into electrospun polymer fibers yields biocompatible and antibacterial mats for potential dressings with improved wound-healing propertie…
Alginate/silica composite hydrogel as a potential morphogenetically active scaffold for three-dimensional tissue engineering
Pursuing our aim to develop a biomimetic synthetic scaffold suitable for tissue engineering, we embedded bone cells, osteoblast-related SaOS-2 cells and osteoclast-like RAW 264.7 cells, into beads, formed of a Na-alginate-based or a silica-containing Na-alginate-based hydrogel matrix. The beads were incubated either separately (only one cell line in a culture dish) or co-incubated (SaOS-2-containing beads and RAW 264.7 beads). The alginate and alginate/silica hydrogel matrices were found not to impair the viability of the encapsulated cells. In these matrices the SaOS-2 cells retain their capacity to synthesize hydroxyapatite crystallites. The mechanical properties, including surface roughn…
Physicochemical and functional characterization of the polymerization process of the Geodia cydonium lectin
The extracellularly localized, galactose-specific lectin from the sponge Geodia cydonium binds at one class of sites, 40 mol Ca2+/mol lectin with an association constant (Ka) of 0.3 X 10(6)M-1. Stoichiometric calculations reveal that in the extracellular milieu 22 mol Ca2+ (maximum) are complexed per mol lectin. Binding of Ca2+ to the lectin increases its apparent Mr from 44000 to 56000 (electrophoretic determination) or from 36500 to 53500 (high-pressure liquid gel chromatographical determination); the s20, w increases from 4.3 S to 4.5 S if Ca2+ is added to the lectin. In the presence of Ca2+ the lectin undergoes a conformational change perhaps by expanding the carbohydrate side chains wh…
Molecular markers for germ cell differentiation in the demosponge Suberites domuncula
Sponges (phylum Porifera) are simple metazoans for which no molecular information on gametogenesis and larval development is available. To support the current study, it was confirmed by histology that oocytes and larvae were produced by the demosponge Suberites domuncula. Three genes/expressed products from S. domuncula whose expression correlated with sexual reproduction were identified and characterized (they are used here as marker genes): i) a receptor tyrosine kinase (RTK) with sequence similarity in the tyrosine kinase domain to fibroblast growth factor receptors; ii) the sex-determining protein FEM1 and iii) the sperm associated antigen (SAA) of triploblasts. Antibodies against the e…