Aurelia aurita (Cnidaria) oocytes' contact plate structure and development.

One of the A. aurita medusa main mesoglea polypeptides, mesoglein, has been described previously. Mesoglein belongs to ZP-domain protein family and therefore we focused on A.aurita oogenesis. Antibodies against mesoglein (AB RA47) stain the plate in the place where germinal epithelium contacts oocyte on the paraffin sections. According to its position, we named the structure found the "contact plate". Our main instrument was AB against mesoglein. ZP-domain occupies about half of the whole amino acid sequence of the mesoglein. Immunoblot after SDS-PAGE and AU-PAGE reveals two charged and high M(r) bands among the female gonad germinal epithelium polypeptides. One of the gonads' polypeptides M(r) corresponds to that of mesogleal cells, the other ones' M(r) is higher. The morphological description of contact plate formation is the subject of the current work. Two types of AB RA47 positive granules were observed during progressive oogenesis stages. Granules form the contact plate in mature oocyte. Contact plate of A.aurita oocyte marks its animal pole and resembles Zona Pellucida by the following features: (1) it attracts spermatozoids; (2) the material of the contact plate is synthesized by oocyte and stored in granules; (3) these granules and the contact plate itself contain ZP domain protein(s); (4) contact plate is an extracellular structure made up of fiber bundles similar to those of conventional Zona Pellucida

Fully formed contact plate immunogold stained.

<p>Staining conditions are the same as on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046542#pone-0046542-g005" target="_blank">Fig. 5</a>. a –contact plate of the oocyte VII final stage at low magnification. Indicated with lines at the right are: O – oocyte, CP – contact plate, ge – germinal epithelium. Scale bar –5 µm. a′ – high magnification of the part of the image on a; gold particles pseudocolored yellow. Scale bar–2 µm. b – type 2 granule presumably in the process of excretion (not stained). Scale bar – 2 µm.</p

Fertilization <i>in vitro</i>.

<p>Spermatozoids added to medusa eggs <i>in vitro</i> in sea water. a - phase contrast microscope, squash preparation. N – eggs' nucleus, sp – spermatozoids. Scale bar – 10 µm. b – c′ – whole egg in the process of fertilization (non-squashed); b – DAPI staining in grayscale, low magnification; c and c′ - phase contrast and DAPI fluorescence in grayscale, the same field. Scale bar – 50 µm.</p

Ultrastructure of medusas' growing oocyte.

<p>I, III, IV, VI, VII – stages of medusas' oocyte development; the same structures as on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046542#pone-0046542-g002" target="_blank">fig. 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046542#pone-0046542-g003" target="_blank">fig. 3</a> are indicated and in addition: mt – mitochondrion, Y – yolk granule. Granules are indicated with black arrows and granule type is indicated with 1 and 2 on b (III), c (IV) and d (VI). Germinal epithelium protrusions are indicated by black arrows with an asterisk on c (IV) and e (VI). b′ and b″ represent the high magnification of parts from b (III). Scale bar – 5 µm for a (I), b (III), c (IV), d (VI), e (VI). Scale Bar – 2 µm for b′ (type 2 granule) and b″ (type 1 granule).</p

Electrophoresis and immunoblot of medusa cell types.

<p>I – SDS-PAGE (a) and immunoblot (b). a – Coomassie-stained 10% SDS-PAGE: 1 (Mes) – mesoglea of adult <i>A.aurita</i>; 2 (Ect) – ectodermal tissue layer; 3 (Mc) – mesogleal cells; 4 (Gf) – germinal epithelium of female gonad. <b><i>M_r</i></b> of marker proteins in kDa are indicated on the left. b – immunoblot of the correspondent lines - Mes, Ect, Mc and Gf. II – AU-PAGE (a) and immunoblot (b). a – Coomassie-stained 7% AU-PAGE: 0 – hialuronidase (pI 9.2) marked with asterisk; 1 (Mes) – mesoglea of adult <i>A.aurita</i>; 2 (Ect) – ectodermal tissue layer; 3 (Mc) – mesogleal cells; 4 (Gf) – germinal epithelium of females' gonad. Arrow marks the start of the separating gel. b – immunoblot. For both immunoblots (b) - primary antibodies RA47 in final dilution 1∶5000; secondary antibodies were antirabbit IgG conjugated with alkaline phosphatase in final dilution 1∶20000 (Sigma).</p

Immunostaining on paraffin sections of medusa female s' gonads.

<p>I, IV, VI, VII – development stages of medusa oocyte. First row - phase contrast; second row – immunostaning of the same preparation. Indicated: Mes – adjacent mesoglea, O – oocyte, N – oocytes' nuclei, g – granules in the oocyte; CP – contact plate. For immunostaining RA47 in final dilution 1∶2000; antirabbit AB conjugated with rhodamine in final dilution 1∶200 (Sigma). Scale bar – 10 µm.</p

The exon junction complex factor Y14 is dynamic in the nucleus of the beetle Tribolium castaneum during late oogenesis

Abstract Background The oocyte chromosomes of the red flour beetle, Tribolium castaneum, are gathered into a knot, forming a karyosphere at the diplotene stage of meiotic prophase. Chromatin rearrangement, which is a characteristic feature of oocyte maturation, is well documented. The T. castaneum karyosphere is surrounded by a complex extrachromosomal structure termed the karyosphere capsule. The capsule contains the vast majority of oocyte RNA. We have previously shown using a BrUTP assay that oocyte chromosomes in T. castaneum maintain residual transcription up to the very end of oocyte maturation. Karyosphere transcription requires evidently not only transcription factors but also mRNA processing factors, including the components of the exon junction complex with its core component, the splicing factor Y14. We employed a gene engineering approach with injection of mRNA derived from the Myc-tagged Y14 plasmid-based construct in order to monitor the newly synthesized fusion protein in the oocyte nuclei. Results Our preliminary data have been presented as a brief correspondence elsewhere. Here, we provide a full-length article including immunoelectron-microscopy localization data on Y14–Myc distribution in the nucleus of previtellogenic and vitellogenic oocytes. The injections of the fusion protein Y14–Myc mRNA into the oocytes showed a dynamic pattern of the protein distribution. At the previtellogenic stage, there are two main locations for the protein: SC35 domains (the analogues of interchromatin granule clusters or nuclear speckles) and the karyosphere capsule. At the vitellogenic stage, SC35 domains were devoid of labels, and Y14–Myc was found in the perichromatin region of the karyosphere, presumably at the places of residual transcription. We show that karyosphere formation is accompanied by the movement of a nuclear protein while the residual transcription occurs during genome inactivation. Conclusions Our data indicate that the karyosphere capsule, being a destination site for a protein involved in mRNA splicing and export, is not only a specializes part of nuclear matrix separating the karyosphere from the products of chromosome activity, as believed previously, but represents a special nuclear compartment involved in the processes of gene expression in the case the karyosphere retains residual transcription activity