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First published online August 17, 2004
doi: 10.1242/10.1242/jcs.01315

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Identification of novel Prominin-1/CD133 splice variants with alternative C-termini and their expression in epididymis and testis

¹ Max-Planck-Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany
² Department of Neurobiology, University of Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany
³ Medical Clinic and Polyclinic I, Technical University Dresden, Fetscherstrasse 74, 01307 Dresden, Germany

View larger version (24K): [in a new window]   Fig. 1. Identification and characterization of multiple mouse prominin-1 splice variants. (A) Sites of alternative splicing in prominin-1. The C-terminal sequences of the testis-derived splice variants s3, s4, s5 and s6 are distinct from that of the previously described kidney-derived prominin-1.s1. Arrowheads indicate exon boundaries in the prominin-1 gene. In addition, the s4 variant lacks five amino acid residues in the first extracellular domain, and the s4 and s5 variants both lack a facultative exon in the second extracellular domain (gray arrows, sequences in parentheses). Forks indicate the potential N-glycosylation sites. (B) Genomic organization of part of the murine prominin-1 gene. Exons 24-26 appear as boxes and introns as solid lines. Exons 25a and 25b are in red and green, respectively. The position of the fifth transmembrane domain (TM5) is indicated in black. Arrows mark the position of the prominin-1-specific primers S-TM5 (left) and AS-exon 26 (right) used for PCR analysis. (C) Distinct prominin-1 mRNAs are generated by the use of alternative acceptor sites (s3-s5) and by intron retention (s6). PCR reactions were performed using first-strand cDNA prepared from mouse kidney or testis as template, and the primers indicated in (B). Arrows indicate the three major PCR products and the corresponding prominin-1 mRNA species. , the 3' end of the open reading frame.

View larger version (67K): [in a new window]   Fig. 2. Tissue distribution of mouse prominin-1 splice variant mRNAs. (A,B) Northern blot of poly(A)+ RNA (2 µg each) from various mouse tissues, analyzed first with cDNA probe E2 (A) and then with a probe for the 3'-UTR (B). (A) The membrane was exposed for 1 day (right) and 3 days (left). (B) The membrane was exposed for 3 days. RNA size markers are indicated on the left (A). (C) PCR reactions were performed using first-strand cDNA prepared from mouse tissues as template using S-TM5 and AS-T1 primers located in exons 24 and 25a, respectively. Prominin-1.s3 and s6 variant cDNAs were used as templates for positive controls (lanes s3 and s6). As a negative control, template was omitted (lane H2O). PCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was performed as a quality control. Embryonic stages (7- to 17-day-old embryos) are indicated as E7 to E17.

View larger version (30K): [in a new window]   Fig. 3. Characterization of prominin-1 splice variants in the adult murine male reproductive system. (A) Proteins solubilized from adult mouse testis and epididymis membranes (T+E, 80 µg protein), testis membranes (T, 80 µg protein) and epididymis membranes (E, 40 µg protein) were incubated in the absence (CONTROL) or presence (PNGase F) of PNGase F, and analyzed by immunoblotting using either mAb 13A4 (13A4, left) or I3 antiserum (I3, right). For comparison, prominin-1 from adult kidney membranes (K, 50 µg protein) was analyzed in parallel. Arrowhead, glycosylated 100-kDa form of testis-derived prominin-1; bracket, glycosylated 104-112 kDa forms of epididymis-derived prominin-1; open arrows, N-deglycosylated, 13A4- and I3-immunoreactive 92-kDa form of epididymis-derived prominin-1; solid arrow, N-deglycosylated, 13A4-immunoreactive, I3-nonreactive 89-kDa form of testis/epididymis-derived prominin-1. (B) Lysates prepared from CHO cells transfected with prominin-1 splice variants s1, s3, s4, s5 or s6, or with vector DNA without insert (M), and, for comparison, prominin-1 from adult kidney (K) and epididymis (E) membranes, were incubated in the absence (top, middle) or presence (bottom) of PNGase F and analyzed by immunoblotting. (Top) Asterisks, endo-H-sensitive forms of recombinant prominin-1; dots, endo-H-resistant forms of recombinant prominin-1. (Bottom) Arrowhead, N-deglycosylated 94-kDa form of kidney-derived prominin-1; open arrow, N-deglycosylated 92-kDa form of epididymis-derived prominin-1; solid arrows, N-deglycosylated 89-kDa form of epididymis-derived prominin-1; diamond, N-deglycosylated 90-kDa form of recombinant s3 variant. Notice that the 89-kDa form of epididymis-derived prominin-1 and recombinant s6 variant exhibit an indistinguishable electrophoretic mobility upon N-deglycosylation, whereas the recombinant s3 variant shows a slightly lower electrophoretic mobility (90 kDa).

View larger version (64K): [in a new window]   Fig. 4. Cell surface expression and localization in plasma-membrane protrusions of prominin-1.s3 and s6 splice variants in CHO cells. (A,B) CHO cells stably transfected with prominin-1 splice variants s1 (positive control), s3, s4, s5, s6 or with vector DNA without insert (MOCK) were either double cell-surface labeled with the mAb 13A4 and FITC-conjugated wheat-germ agglutinin (WGA) (green) followed by Cy3-conjugated anti-rat secondary antibody (red) (13A4) (A) or labeled after paraformaldehyde fixation and Triton-X-100 permeabilization with mAb 13A4 followed by Cy3-conjugated anti-rat secondary antibody (red) (B). (A) Single optical sections at the level of the middle of the cells. (B) Composite pictures of eight optical sections. Scale bars, 45 µm (A), 60 µm (B). (C) Ultrathin cryosections of CHO cells transfected with s3 and s6 splice variants were stained with either the mAb 13A4 (s3) or E3 antiserum (s6) followed by rabbit anti-rat IgG/IgM and/or 15-nm Protein-A/gold. Scale bars, 245 nm (s3); 420 nm (s6).

View larger version (96K): [in a new window]   Fig. 5. Immunohistochemistry showing the regional distribution of prominin-1 in murine epididymis. (A) Histology of adult murine epididymis stained with toluidine blue showing the ductuli efferentes (de), initial segment (is), distal caput (dc) and part of the corpus (c). (B,C) Transverse cryosections of adult murine epididymis were double immunolabeled with mAb 13A4 (13A4) and I3 antiserum (I3) followed by Cy3-conjugated anti-rat and Cy2-conjugated anti-rabbit secondary antibodies. Nuclei were labeled with Hoechst dye. Asterisks mark spermatozoa. Arrows indicate the apical surface of the cauda epithelium without prominin-1 immunostaining. All fluorescence images are at the same magnification. Scale bars, 100 µm. (D) Cryosections of cauda were immunolabeled with mAb 13A4 followed by peroxidase-coupled goat anti-rat, and observed using Nomarski optics. Asterisks mark spermatozoa. Boxes indicate the apical surface of the cauda epithelium without prominin-1 immunostaining. Scale bar, 40 µm.

View larger version (199K): [in a new window]   Fig. 6. Immunoperoxidase localization of prominin-1 in the tail of spermatozoa found in seminiferous tubules. Cryosections of adult murine testis (A,B) and epididymis (C) were immunolabeled with (A-C) or without (A'-C') mAb 13A4 followed by peroxidase-coupled goat anti-rat, and observed using Nomarski optics. (A) Arrows indicate the lumen of seminiferous tubules. (B) The asterisk indicates the tail of spermatozoa, shown at high magnification in the inset. (C) Arrowheads indicate the apical surface of the corpus epithelium. Scale bars, 170 µm (A,A'); 30 µm (B,B'); 40 µm (C,C').

View larger version (107K): [in a new window]   Fig. 7. Immunogold electron microscopy showing the subcellular localization of prominin-1 in murine epididymis. Ultrathin cryosections of the apical surface of epithelial cells lining the corpus of the epididymis were incubated with (A) or without (B) E3 antiserum followed by 15-nm Protein-A/gold. Arrows indicate regions of the plasma membrane between the stereocilia; asterisks indicate spermatozoa. Scale bars, 625 nm (A); 640 nm (B).

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