Effect of cisplatin and doxorubicin on primordial follicles, and on expression of cleaved PARP.

<p>(<b>A, B</b>)<b>:</b> Analysis of TUNEL-positive cells within primordial follicles. (<b>A</b>)<b>:</b> Total number of primordial follicles, and number of primordial follicles containing TUNEL-positive cells, in ovaries treated with cisplatin or doxorubicin. (<b>B</b>)<b>:</b> TUNEL-positive primordial follicles further categorised into percentage in which the oocyte or the granulosa cells stained positive. Bars denote mean+sem; n = 4–5; stars denote significant differences relative to control (**P<0.01). (<b>C, D</b>)<b>:</b> Cleaved PARP expression in cisplatin and doxorubicin treated ovaries. Protein expression of cleaved PARP relative to β actin (loading control) in whole newborn ovaries following 24 h of (<b>C</b>)<b>:</b> cisplatin or (<b>D</b>)<b>:</b> doxorubicin treatment. Examples of Western blots are shown in Supporting Information, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070117#pone.0070117.s003" target="_blank">Figure S3</a>. Bars denote mean+sem; n = 3; stars denote significant differences relative to control (*p<0.05, **p<0.01, ***p<0.001).</p

Cisplatin and doxorubicin both lead to loss of follicle health and a reduction in follicle numbers.

<p>(<b>A</b>) Cisplatin; (<b>B</b>) Doxorubicin: (<b>i</b>) Percentage of unhealthy follicles (clear); and (<b>ii</b>) total number of follicles (shaded) in each ovary. Bars denote mean+sem; n = 5 for all groups, stars denote significant differences relative to control (*p<0.05, **p<0.01, ***p<0.001).</p

Cisplatin and doxorubicin affect different follicle classes.

<p>Follicles were classified as morphologically unhealthy in cisplatin and doxorubicin treated ovaries according to follicle type. Effect of (<b>A</b>) Cisplatin or (<b>B</b>) Doxorubicin on the percentage of transitional and primary follicles classified as morphologically unhealthy. Bars denote mean+sem; n = 5 for all groups, stars denote significant differences relative to control (*p<0.05, **p<0.01, ***p<0.001).</p

High Fat/High Fructose diet effects on <i>Gys1</i>, <i>Gsk3β</i> and <i>Akt1</i> in hippocampus and cortex.

<p>Open bar, control diet; solid bar, control diet plus cinnamon, mottled gray bar, HF/HFr diet; gray bar, HF/HFr diet plus cinnamon. Values are mean ± SE for 8 to 10 rats. Different letters denote significant differences among groups, p<0.05.</p

Cisplatin and doxorubicin affect different follicular cell types.

<p>Ovaries were treated with Cisplatin or Doxorubicin. All unhealthy transitional and primary follicles were further categorized as unhealthy due to: (<b>A</b>) poor oocyte health; (<b>B</b>) poor granulosa cell health; or (<b>C</b>) both. Bars denote mean+sem; n = 5 for all groups, stars denote significant differences relative to control (*p<0.05, **p<0.01, ***p<0.001).</p

Representative histological sections of cultured mouse ovaries.

<p>(<b>A, B</b>)<b>:</b> TUNEL-analysis to determine apoptosis in primordial follicles. (<b>A</b>)<b>:</b> ovary section labelled with TUNEL reaction (green) and counterstained with DAPI (blue). Inset top- and bottom-right magnification images are of the respective framed areas, illustrating examples of TUNEL positive oocytes (white arrowheads) and TUNEL positive granulosa cell (white arrow) within follicles identified as at the primordial stage in (B). (<b>B</b>)<b>:</b> section in (A) subsequently stained with haematoxylin and eosin. Inset sections in (A) correspond here to degenerated oocytes (black arrowheads) and degenerated surrounding granulosa cells (black arrow) within primordial follicles. Scale bar = 50 µm. (<b>C–H</b>)<b>:</b> Photomicrographs of haemotoxylin and eosin stained sections from ovaries treated with (<b>C</b>) control, (<b>D</b>) 3 µg ml⁻¹ imatinib, (<b>E</b>) 0.5 µg ml⁻¹ cisplatin, (<b>F</b>) cisplatin and imatinib co-treatment, (<b>G</b>) 0.05 µg ml⁻¹ doxorubicin and (<b>H</b>) doxorubicin and imatinib co-treatment. Scale bars represent 25 µm. Examples of a healthy primordial follicle (arrow), healthy growing follicle (black arrowhead) and unhealthy growing follicle (white arrowhead) are shown.</p

Imatinib co-treatment with cisplatin, but not doxorubicin, rescues follicle health.

<p>Control, cisplatin-treated (0.5 µg ml⁻¹) and doxorubicin-treated (0.05 µg ml⁻¹) cultured ovaries were cultured in the presence or absence of imatinib. (<b>A</b>)<b>:</b> Percentage of unhealthy follicles (clear); (<b>B</b>)<b>:</b> Total number of follicles (shaded). Bars denote mean+sem; n = 7 for all groups, stars denote significant differences relative to control (**p<0.01). (<b>C</b>)<b>:</b> Pathway by which imatinib could protect the ovary against the damaging effect of cisplatin more effectively than it could against the damaging effect of doxorubicin.</p

High Fat/High Fructose diet increases <i>Insr</i>, <i>Irs1</i> and <i>Irs2</i> in hippocampus and cortex.

<p>Open bar, control diet; solid bar, control diet plus cinnamon, mottled gray bar, HF/HFr diet; gray bar, HF/HFr diet plus cinnamon. Values are mean ± SE for 8 to 10 rats. Different letters denote significant differences among groups, p<0.05.</p

High Fat/High Fructose (HF/HFr) diet decreases <i>Glut 1</i> and 3 in hippocampus and cortex.

<p>Open bar, control diet; solid bar, control diet plus cinnamon, mottled gray bar, HF/HFr diet; gray bar, HF/HFr diet plus cinnamon. Values are mean ± SE for 8 to 10 rats. Different letters denote significant differences among groups, p<0.05.</p

High Fat/High Fructose diet increases <i>Pten</i>, <i>Tau</i> and <i>App</i> in hippocampus and cortex.

<p>Open bar, control diet; solid bar, control diet plus cinnamon, mottled gray bar, HF/HFr diet; gray bar, HF/HFr diet plus cinnamon. Values are mean ± SE for 8 to 10 rats. Different letters denote significant differences among groups, p<0.05.</p