Effects of E2, PPT, DPN, ZYC-26 and ZYC-23 on intracellular lipid peroxidation in BSO-treated FRDA fibroblasts.

<p>All steroid concentrations were 100 nM, DMSO concentration was 0.1% and BSO concentration was 1 mM. Depicted are mean ± SD for n = 8 per group. * indicated p<0.05 versus BSO alone-treated cells. 1.0 normalized 8-isoprostane control concentration = 6.23 pg/mL.</p

Proposed mechanism of 17β-Estradiol in BSO-treated FRDA fibroblasts.

<p>Proposed mechanism of 17β-Estradiol in BSO-treated FRDA fibroblasts.</p

Effects of E2, PPT, DPN, ZYC-26 and ZYC-23 on the activity of aconitase in BSO-treated FRDA fibroblasts.

<p>All steroid concentrations were 100 nM, DMSO concentration was 0.1% and BSO concentration was 1 mM. Depicted are mean ± SD for n = 8 per group. * indicated p<0.05 versus BSO alone-treated cells.</p

Effects of E2, PPT, DPN, ZYC-26 and ZYC-23 on the collapse of mitochondrial membrane in BSO-treated FRDA fibroblasts.

<p>All steroid concentrations were 100 nM, DMSO concentration was 0.1% and BSO concentration was 1 mM. Depicted are mean ± SD for n = 8 per group. * indicated p<0.05 versus BSO alone-treated cells.</p

Effects of E2 and ZYC-26 on mitochondrial function in BSO-treated FRDA fibroblasts.

<p>A.) Oxygen consumption rate (OCR; in pMoles/min) B.) Basal respiratory rate (in pMoles/min) C.) Maximal respiratory rate (in pMoles/min) All steroid concentrations were 100 nM, DMSO concentration was 0.1% and BSO concentration was 1 mM. Depicted are mean ± SD for n = 8 per group. * indicated p<0.05 versus BSO alone-treated cells.</p

Figure 3

<p>A.) Calcein AM imaging demonstrating cell viability between vehicle control and BSO treatment groups at 24, 36 and 48 hours. Scale bar = 200 µm. B.) Effects of E2, PPT, DPN, ZYC-26 and ZYC-23 on cell viability in BSO-treated FRDA fibroblasts. All steroid concentrations were 100 nM, DMSO concentration was 0.1% and BSO concentration was 1 mM. Depicted are mean ± SD for n = 8 per group. * indicated p<0.05 versus BSO alone-treated cells.</p

Structures of compounds assessed for protection against BSO toxicity in FRDA fibroblasts.

<p>Structures of compounds assessed for protection against BSO toxicity in FRDA fibroblasts.</p

Maximal Donor Germline Transmission from DAZL-Deficient Rats.

<p>(A) Southern blot analysis of progeny from Wildtype (WT) and DAZL-Deficient (Dazl) recipient rats transplanted with 50,000 <i>GCS-EGFP</i> spermatogonia/right testis at passage 13 (i.e. 158 days in culture); left testes of each animal were not transplanted. At 75 days post-transplantation recipients (R) were paired with WT females (F) and allowed to produce pups by natural breeding (See R942-R949 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006308#pone-0006308-t001" target="_blank">Table 1</a>). Shown are blots from representative litters probed for <i>EGFP</i> to distinguish progeny produced by donor cells. OMP = loading control. Genomic DNA Controls were from untreated <i>GCS-EGFP</i> and <i>DAZL</i>-Deficient transgenic rats. LTR = PCR primers specific for lentiviral transgene in <i>DAZL</i>-deficient rats. GAPDH = PCR loading control. (B) Bright field and green fluorescence images of testes from Wildtype (<i>Left</i>) and DAZL-deficient (<i>Right</i>) recipient rats at 212 days post-transplantation. Scale bar = 1 cm. (C) Graph of germline transmission rates for the donor, <i>GCS-EGFP</i> transgene from Wildtype and DAZL-deficient recipient rats transplanted with 50,000 <i>GCS-EGFP</i> spermatogonia/right testis at passage 13; left testes were not transplanted. <i>DAZL</i>-deficient recipients transmitted the <i>GCS-EGFP</i> transgene to 100%+/−0% of progeny (+/−SEM, n = 3 recipients; 9 litters), with 73 of 73 total F1 pups born from donor cells. Wildtype recipients transmitted the <i>GCS-EGFP</i> transgene to 14%+/−5.9% of progeny (+/−SEM, n = 3 recipients; 9 litters), with 16 of 116 total F1 pups born from donor cells. (D) Genealogy tree showing stable transmission of donor haplotypes from <i>DAZL</i>-Deficient recipients (F0) R989 and R990 to F1 and F2 progeny. Recipients were each transplanted with 150,000 rat spermatogonia/testis from line RSGL-GCS9 at passage 17 (See R988-R990 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006308#pone-0006308-t001" target="_blank">Table 1</a>). Spermatogonial line RSGL-GCS9 was derived from a rat homozygous for the <i>GCS-EGFP</i> transgene <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006308#pone.0006308-Wu1" target="_blank">[36]</a>. Thus, F1 progeny represent half-siblings; some of which were crossed to re-derive transgenic F2 progeny homozygous for the <i>tgGCS-EGFP</i> allele.</p

Effects of E2, PPT, DPN, ZYC-26 and ZYC-23 on the intracellular ATP concentration inside of BSO-treated FRDA fibroblasts.

<p>All steroid concentrations were 100 nM, DMSO concentration was 0.1% and BSO concentration was 1 mM. Depicted are mean ± SD for n = 8 per group. * indicated p<0.05 versus BSO alone-treated cells. 100% normalized ATP control concentration = 501 pM.</p

Long-Term Spermatogenic Potential of Donor Spermatogonia.

<p>(<i>Left</i>) Graph showing relative numbers of Round and Elongating Spermatids in seminiferous tubules of non-transplanted, non-busulfan-treated, Wildtype and <i>DAZL</i>-deficient rat lines at 4 months of age (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006308#pone-0006308-g001" target="_blank">Fig. 1a</a>), in comparison to Spermatid numbers in busulfan-treated, <i>DAZL</i>-deficient recipient rats at 212 days (i.e. ∼8 months of age) after being transplanted with rat spermatogonial line, RSGL-GCS9, at passage 13 (i.e. culture day 158). Cell counts were normalized/1000 Sertoli cells. +/−SEM, n = 3 rats/group. (<i>Right</i>) Images of histological sections of seminiferous tubules from the <i>DAZL</i>-deficient recipient rats described in the “Left” panel after being transplanted with spermatogonia from RSGL-GCS9. <i>Bottom Right</i> shows a higher magnification image within the boxed region of the <i>Top Right</i> panel. Scale bars = 100 µm.</p