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First published online 17 December 2003
doi: 10.1242/dev.00918

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`Side Population' cells in adult mouse testis express Bcrp1 gene and are enriched in spermatogonia and germinal stem cells

¹ Laboratoire de Méiose et de Maturation Gamétique, DRR / DSV/ CEA – U566 INSERM – Université Paris 7, Fontenay aux Roses, France
² Laboratoire de Radiosensibilité des Cellules Germinales, DRR / DSV/ CEA – U566 INSERM – Université Paris 7, Fontenay aux Roses, France
³ CNRS FRE 2358, Génétique Expérimentale et Moléculaire, Orléans, France

View larger version (50K): [in a new window]   Fig. 1. Flow cytometric analysis of Hoechst 33342 fluorescence. (A) C57BL/6 adult murine testicular cells. (B) Definition of the five main subpopulations and respective percentages of total testicular viable cells: 1 (1%), 2 (2.4%), 3 (8%), 4 (7%), 5 (70%). (C,D) Comparison with two infertile murine models: W/Wv mutant (C) and cryptorchid C57BL/6 male (D). (E,F) Identification of somatic interstitial cells: interstitial cell-enriched fraction (E) compared with the fraction enriched in seminiferous tubules (F). Vertical axis shows blue (Ho) fluorescence, horizontal axis red (Ho/PI) fluorescence.

View larger version (51K): [in a new window]   Fig. 2. Analysis of differentiation marker expression on the sorted cell subpopulations. RNA from whole testis cell suspension of mutant W/Wv males was analyzed in parallel with a sorted cell population (according to Fig. 1B) from normal adult males: population 1 (SP), population 3 (4n), upper diploid population 4 (2n) and population 5 (n). RT-PCR was performed for Kit (35 cycles), Dazl (30 cycles), H1t (30 cycles), Hsp70-2 (30 cycles) and Crem (30 cycles) genes. ß-actin (30 cycles) served as a normalization control.

View larger version (45K): [in a new window]   Fig. 3. Analysis of the SP population for the expression of 6-integrin and Stra8 germinal stem cell markers. Ho fluorescence flow cytometric analyses of 6-integrin-positive cell fraction (A) and 6-integrin-negative cell fraction (B) after immunomagnetic sorting. (C) RT-PCR analysis of Stra8 gene expression (35 cycles) on W/Wv, and sorted normal populations: SP cells (SP), tetraploid population (4n), upper diploid population (2n) and haploid population (n).

View larger version (33K): [in a new window]   Fig. 4. The germinal SP phenotype is related to ABC transporter activity. 6-integrin-positive cell fraction stained with Ho without inhibitor (A), with deoxyglucose plus sodium azide (B), 25 µg/ml (C) or 75 µg/ml of verapamil (D). (E) Bcrp1 expression analysis (35 cycles) in testicular subpopulations: SP cells (SP), tetraploid population (4n), upper diploid population (2n) and haploid population (n). 6-integrin-positive cell fraction stained with Ho without inhibitor (F) and with specific BCRP1 inhibitor Ko143 (G).

View larger version (52K): [in a new window]   Fig. 5. In vivo repopulation assay of the testis SP EGFP-positive in busulfan-treated recipient analysed 10 weeks after transplantation. (A) Ho fluorescence flow cytometric analyses of 6-integrin-positive cell fraction from EGFP donor cells. Sorting gate of the testis SP is enclosed in box. (B) Detection of fluorescent seminiferous tubules in a recipient testis transplanted with the EGFP testis SP as determined by fluorescence microscopic analysis of whole testis. (C) Flow cytometric analysis of EGFP and Ho blue fluorescences of testicular single cell suspensions prepared from recipient testis transplanted with the EGFP testis SP and (D) Ho fluorescence flow cytometric (red/blue Ho fluorescence) analysis of EGFP-positive cells in recipient testis gated from Fig. 5C. (E) Green and red fluorescence analysis of testicular cell suspension from recipient testis in order to determine EGFP-positive cell number per recipient testis by flow cytometry using TruCountTM (BD Biosciences) methodology (see Materials and methods). EGFP-positive cells (EGFP) and TruCount fluorescent beads (FB) are boxed. (F) Enhanced colonization of recipient testis by the transplanted testis SP (n=8 testis) compared with total population (n=8 testis). EGFP-positive cell number per recipient testis was normalized to 105 cells injected. The values are mean±s.e.m. Difference between SP and total cells is significant (P<0.03). Results were obtained from three independent experiments.