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

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

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