Whole mount preparation, Nomarski optics. dipterans, the associates of various phylogenetic lineages may show significant variations from the type (Kubrakiewicz et al. 2003; Mazurkiewicz and Kubrakiewicz 2005; Tworzydlo et al. 2005; Jaglarz et al. 2009, 2010). Follicular epithelium differentiation in Lepidoptera has been studied in several varieties: (King and Aggarwal 1972), (Cummings 1972; Cruickshank 1973; Torres 1981), (Yamauchi and Yoshitake a, b) but data from these investigations are fragmentary and concentrate Rabbit Polyclonal to CELSR3 primarily within the contribution of the follicular cells in vitellogenesis and eggshell formation (Kawaguchi et al. 1996, 2000; Sarto et al. 2005; Candan et al. 2008). With this paper, we provide the 1st detailed description of the follicular epithelium differentiation and Benzylpenicillin potassium diversification in butterflies. Materials and methods With this paper, we used polytrophic ovaries of were collected in SW Poland in the period 2008C2010. Preparation of whole mounts The ovaries were dissected and fixed for 40?min in 4% formaldehyde in phosphate-buffered saline PBS (NaCl, 137?mM; KCl, 2.7?mM; Na2HPO4, 8?mM; KH2PO4, 1.5?mM) containing 0.1% Triton X-100. After a few rinses with PBS, the material was first examined having a stereomicroscope Olympus SZX 10 and a light microscope equipped with Nomarski optics and then subjected to whole-mount fluorescent staining. For detection of cell nuclei (DNA), the material was stained with 0.2?mg/ml DAPI (4,6 diamidino-2-phenylindole dihydrochloride) (Sigma, D9542) for 20?min in darkness. For detection of microfilaments (F-actin), the ovaries were stained with 2?mg/ml rhodamine-conjugated phalloidin (Sigma, P1951) for 20?min in darkness. In both cases, after rinsing with buffer, the ovarioles were whole-mounted onto microscope slides and examined with either an Olympus BHS light microscope equipped with an epifluorescence device or with an Olympus FV1000 confocal microscope. Histological and ultrastructural analysis Ovaries were dissected and fixed at RT in 2.5% glutaraldehyde in 0.1?M phosphate buffer (pH?=?7.4) for a few weeks. The material was rinsed several times with phosphate buffer and postfixed in a mixture comprising 1% osmium tetroxide and 0.8% potassium ferrocyanide for 1?h (according to McDonald, 1984). After dehydration inside a graded series of acetone, the material was inlayed in Benzylpenicillin potassium Epon 812 (Serva, Heidelberg, Germany). Semithin sections (0.6?m solid) were stained with 1% methylene blue and examined with the Olympus BHS microscope. Ultrathin sections were contrasted with uranyl acetate and lead citrate according to the standard methods and examined having a Zeiss EM 900 electron microscope at 80?kV. Results Morphology of the ovary Each of the combined ovaries of is composed of four long ovarioles of meroistic polytrophic type (Fig.?(Fig.1a).1a). Individual ovarioles are covered by a relatively solid ovariolar sheath and a coating of muscle Benzylpenicillin potassium tissue (Fig. 2a, cCe). Each ovariole is built of four linearly arranged parts: terminal filament, germarium, vitellarium, and ovariolar stalk. Terminal filaments join up with each other and form a ligament that attaches the gonad to the body wall. Open in a separate windowpane Fig. 1 Morphology of the ovariole. a The ovariole consists of terminal filament (TF), germarium (G), and vitellarium (V). In vitellarium, several egg chambers in consecutive phases of oogenesis are arranged linearly. (A-P) refers to anterior-posterior axis of the ovariole. Stereomicroscope. Whole mount preparation. Level pub?=?1?mm. b The part Benzylpenicillin potassium of germarium with zones III and IV. In zone III, degenerating cells (d) are visible. Zone IV is definitely filled with cystocytes in 1st meiotic prophase (Cc). Arrows show prefollicular cells in the peripheral parts of the ovariole. ArrowheadCovariolar sheath. Semithin section after methylene blue. Level pub?=?40?m. c The part of vitellarium with egg chambers in early previtellogenic phases. Nurse cells (NC) occupy the anterior part of the egg chamber, while the oocyte (Oo) is located in its posterior part. In the nurse cell nuclei (n), patches of dense material are visible. Relatively large oocyte nucleus (N) occupies a central position in the ooplasm. mbFC, mainbody follicular cells; stFC, stretched follicular cells. Hollow arrow shows nuclear body in the oocyte karyoplasm. Semithin section after methylene blue. Level pub?=?40?m. d The part of vitellarium with egg chambers in advanced previtellogenic phases. Oocyte (Oo) nucleus (N) is visible on either part of the ooplasm. FC, follicular cells; NC, nurse cells. (A-P) refers to anterior-posterior axis of the ovariole. Whole mount preparation, Nomarski optics. Level pub?=?50?m Open in a separate windowpane Fig. 2 Previtellogenesis. Early stages of follicular epithelium differentiation. a Part of germarium:zones III and IV. Clusters of cystocytes (Cc) are surrounded by.