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First published online 15 March 2006
doi: 10.1242/dev.02303

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FGF9 promotes survival of germ cells in the fetal testis

Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA

View larger version (30K): [in a new window]   Fig. 1. Analysis of germ cell loss in Fgf9–/– gonads. (A) Alkaline phosphatase staining for germ cells in Fgf9 mutant gonads at 12.5 dpc. XY Fgf9–/– gonads show a severe reduction in germ cell numbers relative to siblings. (B) Quantification of germ cell loss. Germ cell numbers are similar in mutants and controls from 7.5 to 11.5 dpc (11.5 dpc shown). At 12.5 dpc XY Fgf9–/– gonads contain significantly fewer germ cells than XY Fgf9+/–, XX Fgf9+/– or XX Fgf9–/– (n=14 for each timepoint; P<0.01). Scale bar: 250 µm.

View larger version (107K): [in a new window]   Fig. 2. XY Fgf9–/– gonads show elevated levels of germ cell death between 11.5 and 12.5 dpc. Germ cells are stained for E-Cadherin (blue), whereas laminin (green) labels the basal lamina of testis cords. Cell death is labeled by LysoTracker Red DND-99 (red). XX and XY Fgf9+/– gonads show low basal levels of cell death at both 11.5 dpc (A,G), 12.0 dpc (B,H) and 12.5 dpc (C,I). XX Fgf9–/– gonads contain similar low levels at 11.5 dpc (D), 12.0 dpc (E) and 12.5 dpc (F). XY Fgf9–/– gonads show significantly increased levels of dying germ cells at stages 11.5 to 12.5 dpc (J-L; P<0.05. g, gonad; m, mesonephros. Broken line indicates boundary of gonad and mesonephros. Figures are representative of three independent experiments of n3 each. Scale bar: 50 µm.

View larger version (14K): [in a new window]   Fig. 3. XY Fgf9–/– gonads initiate male development. Quantitative PCR (QPCR) of Sry in 11.5 dpc XX and XY Fgf9+/– and–/– gonads. XY Fgf9–/– gonads express Sry at a similar level to XY Fgf9+/– gonads. n=4 for XX Fgf9+/+, +/– and –/–. n=4 for XY Fgf9+/+ and +/–. n=3 for XY Fgf9–/–.

View larger version (58K): [in a new window]   Fig. 4. 12.5 dpc XY Fgf9 null gonads express markers of ovarian development. In situ hybridization using markers of ovarian fate Bmp2 (A,C,E,G) and follistatin (B,D,F,H) on 12.5 dpc XX (A-D) and XY (E-H) gonads of either Fgf9+/– (A,B,E,F) or Fgf9–/– (C,D,G,H) genotypes. All XX gonads express Bmp2 and follistatin normally (A-D). XY Fgf9+/– samples do not express either of these ovarian markers (E,F). At 12.5 dpc, XY Fgf9–/– gonads express both Bmp2 and follistatin (G,H) in a pattern similar to XX gonads. Sections of 14.5 dpc XX and XY gonads containing germ cells immunostained for PECAM (green) and phosphorylated H2AX (red) (I-K). Virtually all XX Fgf9+/– germ cells are positive for phosphorylated H2AX staining, as expected (I), whereas XY Fgf9+/– germ cells are not (J). Forty-seven percent of the germ cells that survive to this stage in XY Fgf9–/– gonads are positive for phosphorylated H2AX (arrowhead in K), but some are not (arrow in K). Figures representative of three independent experiments of n5 (A-H) each and n=5 (I-K) each. Scale bar: 250 µm in A-H; 25 µm in I,K.

View larger version (73K): [in a new window]   Fig. 5. Time-dependent exposure to culture medium with or without exogenous FGF9 reveals distinct effects of FGF9 on Sertoli and germ cells. Cultured gonads were labeled with antibodies against SOX9 to detect Sertoli cells (green) and PECAM to detect germ cells and vasculature (red). Blue and purple arrows indicate stages at which samples were placed into culture. (A) Uncultured XY Fgf9–/– control with few germ cells, no SOX9-positive cells and no male-specific vasculature. (B) Fgf9+/– 12.5 dpc XY gonad cultured without FGF9 for comparison. (C) XY Fgf9–/– gonad cultured without FGF9 from 11.25 to 12.5 dpc shows few germ cells and many SOX9-positive cells. Some male structures are rescued including testis cord-like organization (arrow) and male-specific vasculature (arrowheads); however, germ cell numbers are not rescued compared with Fgf9+/– (B). (D) When 25 ng/ml FGF9 is added to the culture medium of Fgf9–/– gonads explanted at 11.25 dpc and cultured to 12.5 dpc, germ cells are rescued as well as testis cord formation (arrow) and male-specific vasculature (arrowhead). (E) XY Fgf9–/– gonad cultured from 11.75 to 12.5 dpc shows few germ cells, few SOX9-positive cells and no male-specific vasculature or cord formation. (F) XY Fgf9–/– gonad cultured from 11.75 to 12.5 dpc in the presence of 25 ng/ml FGF9. Germ cell numbers are rescued but other features of testis development are not. SOX9-positive cells are reduced or absent. Figures representative of three independent experiments, n5 each. Scale bar: 50 µm.

View larger version (42K): [in a new window]   Fig. 6. Purified gonocyte culture with or without FGF9 demonstrates direct pro-survival effect of FGF9 on XY gonocytes. Gonocytes of 10.5 dpc (A) or 11.5 dpc (B) Oct4:GFP or XXSryMyc;Oct4:GFP embryos were purified (>95%) and cultured in DMEM alone or in DMEM + FGF9 (25 ng/ml). (A) XX and XY gonocytes (10.5 dpc) decline rapidly in number regardless of treatment. (B) XX and XY gonocytes (11.5 dpc) in DMEM also decline rapidly as do XX gonocytes in DMEM + FGF9. However, XY gonocytes and XXSryMyc gonocytes in DMEM + FGF9 exhibit significantly improved survival after 18 and 36 hours in culture (n=10 for each time point; P<0.001). (C) Alkaline phosphatase staining of XXSryMyc; Fgf9+/– and XXSryMyc; Fgf9–/– gonads at 11.5 and 12.5 dpc. XXSryMyc; Fgf9–/– gonads at 12.5 dpc show reduced numbers of germ cells (n3 for each sample).

View larger version (25K): [in a new window]   Fig. 7. FGF16, but not FGF2 or FGF7, promotes survival of 11.5 dpc XY gonocytes. Gonocytes of 11.5 dpc Oct4:GFP embryos were purified (>95%) and cultured in DMEM alone or in DMEM + 25 ng/ml FGF2 (A), + 25 ng/ml FGF7 (B), + 25 ng/ml FGF16 (C) or + 40 ng/ml FGF16 (D). FGF2 and FGF7 show no effect on gonocyte survival (A,B). However, XY gonocytes and gonocytes cultured in DMEM + 25 ng/ml FGF16 (C) or + 40 ng/ml FGF16 (D) exhibit significantly improved survival after 36 hours in culture (n=10 for each time point; P<0.01).