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

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

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