QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 21 June 2005
doi: 10.1242/jcs.02430

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gillot, I. Articles by Vidal, F. Search for Related Content PubMed PubMed Citation Articles by Gillot, I. Articles by Vidal, F. Social Bookmarking                         What's this?

Germ cells and fatty acids induce translocation of CD36 scavenger receptor to the plasma membrane of Sertoli cells

¹ INSERM UMR 636, Université de Nice-Sophia Antipolis, Faculty of Sciences – Parc Valrose, 06108 Nice Cedex 2, France
² CCMA, Université de Nice-Sophia Antipolis, Faculty of Sciences – Parc Valrose, 06108 Nice Cedex 2, France
³ Departments of Biochemistry and Howard Hughes Medical Institute, Box 357370, University of Washington, Seattle, WA 98195, USA

View larger version (109K): [in a new window]   Fig. 1. Localization of the CD36 protein in the adult testis. (A,B) Immunofluorescent labelling of CD36 on cryosection of adult mouse testis. CD36 protein is observed at various locations depending on the stage of differentiation of the seminiferous epithelium. The protein is located at the vicinity of rounded and elongated spermatids in the apical part of the tubules or in the basal Sertoli cytoplasm. Immunostaining of CD36 (A, B1, B2, B3) and nuclei staining with DAPI (B4, B5, B6). Bar, 50 µm. (C) Confocal microscopy analysis of anti-CD36 immunofluorescent staining (green) and nucleus staining (DAPI, blue) in the apical zone of the seminiferous epithelium. Labelling is observed around structures without nuclei, evocative of residual bodies (arrowheads). Bar, 10 µm. (D) Western blot analysis of CD36 in adult mouse testis (9 month). Molecular masses are represented on the left (kDa).

View larger version (171K): [in a new window]   Fig. 2. Subcellular localization of CD36 in adult testis. Post-embedding immunoelectron microscopy. Ultrathin section were labelled with anti-CD36 antibody. (A) Adluminal part of the seminiferous epithelium of an adult mouse (left; bar, 160 nm); es, elongated spermatid; rs, round spermatid. White rectangle is enlarged in right panel (bar, 430 nm). Transversal section of the flagellum (star) in the middle of the electron dense region of the future residual body indicates the position of the elongated spermatid cytoplasm. The cytoplasmic part of the elongated spermatid, corresponding to residual bodies, is specifically labelled by the antibodies (arrowhead). (B) Basal part of the seminiferous epithelium of an adult mouse (left, bar, 235 nm). White rectangle is enlarged in right panel (bar, 400 nm). Some vesicles are specifically labelled by the antibodies (arrowhead).

View larger version (42K): [in a new window]   Fig. 3. CD36 protein is expressed in Sertoli cells. (A) Immunofluorescence analysis of CD36 protein expression in 15P-1 cells. The protein is mainly located in the perinuclear region of 15P-1 cells (n, nucleus) and not at the plasma membrane (arrowhead). Bar, 10 µm. (B) Immunofluorescence analysis in germ cells of CD36 protein expression (left) and the corresponding pictures with nuclear DAPI staining (right). B1, elongated (es) and round (rs) spermatids; B2, spermatocytes. No signal was observed on freshly isolated pre-meiotic or post-meiotic germ cells. Bar, 10 µm.

View larger version (65K): [in a new window]   Fig. 4. Subcellular relocalization of CD 36 in 15P-1 cells in presence of germ cells. (A,B) Immunostaining of 15P-1 cells with anti-CD36 antibodies (A1,B1) and corresponding DNA staining with DAPI (B2) merge with the visible image (A2). Germ cell fraction enriched in elongated spermatids (sp, A) or in residual bodies (rb, B) were able to induce the same relocation of CD36 protein along the plasma membrane cytoplasmic extensions of 15P-1 cells (arrowhead). Bar, 10 µm. (C,D) Immunogold localization of CD36 on ultrathin cryosections of 15P-1 cells co-cultured in presence of total germ cells. Each experiment was carried out at least twice. (C) Gold particles present at the plasma membrane invagination; bar, 80 nm. (D) Overview of a spermatid engulfed by 15P-1 cell. Bar, 1.25 µm. Below, enlargement of the previous white square displays the immunogold localization of CD36 within phagocytotic vacuole (arrowhead). Bar, 0.22 µm. (E) Western blot analysis performed using specific antibodies against CD36. Lane 1, round spermatid; lane 2, mixed germ cells; lane 3, spermatocytes; lane 4, syncitium, lane 5, 15P-1 cells. Molecular masses are represented on the left (kDa).

View larger version (59K): [in a new window]   Fig. 5. Morphological changes in 15P-1 cell shape during co-culture experiments. (A) Scanning electron microscopic observation of 15P-1 cells co-cultured in presence of germ cells. Cytoplasmic extension of a Sertoli cell (arrowhead) wrapping around a germ cell (star). Bar, 7 µm. (B) Four most representative slides from a 3D reconstruction obtained by deconvolution microscopic analysis. 15P-1 cells are labelled with CMTMR fluorophore and germ cells are labelled with CMFDA fluorophore (see Materials and Methods). The germ cell (star) interacting with 15P-1 cells has both colours. Bar, 7 µm.

View larger version (123K): [in a new window]   Fig. 6. Caveolin-2 co-localizes with CD 36 in testis. (A) Immunostaining of 15P-1 cells with caveolin-1 antibodies (left) and corresponding DNA staining with DAPI (right). (B) Immunostaining with caveolin-1 antibodies of 15P-1 cells co-culture with germ cells (left) and corresponding DNA staining with DAPI (right). (C) Immunofluorescent labelling of CD36 (left) and caveolin-2 (red) with DNA staining (right) on cryosection of adult mouse testis. CD36 and caveolin-2 proteins co-localized (arrowhead) at the vicinity of rounded and elongated spermatids in the apical part of the tubules (bar, 25 µm). (D) Overview of the adluminal part of the seminiferous epithelium of an adult mouse (left; bar, 1.6 µm) and enlargement of the white square (right; bar, 300 nm). CD36 and caveolin immunolocalized at the plasma membrane using gold beads of two different sizes (respectively 15 nm and 10 nm, arrowhead).

View larger version (78K): [in a new window]   Fig. 7. Effect of fatty acid treatment on Sertoli cells. Immunostaining with anti-CD36 antibodies performed on 15P-1 cells, after 4 hours C18:2 treatment (A) or after 15 hours C18:2 treatment (B).

View larger version (60K): [in a new window]   Fig. 8. Fatty acid treatment inhibits the phagocytosis of germ cells but not latex beads. Immunostaining with anti-CD36 antibodies performed on (A) 15P-1 cells (s) co-cultured with germ cells (arrowhead); (B) 15P-1 cells (s) co-cultured with germ cells (arrowhead) in presence of C18:2; (C) 15P-1 cells co-cultured with germ cells and latex beads (arrow) in presence of C18:2 (left). Corresponding field with DNA staining (right). (D) 15P-1 cells were cultured with germ cells (GC) or latex beads (LB) in presence or in absence of fatty acids (FA). After 8 hours co-culture, cells were gently washed with PBS, fixed in 4% PFA and phagocytosed cells or beads were counted. Results were expressed as the proportion (%) of phagocytosed germ cells or latex beads compared with total germ cells or latex beads remaining after washing and fixation. Mean of at least five experiments, bar shows s.e.m. ***: significant variation between GC and GC+FA, P<10–6 (Student's t-test).

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter