Bortezomib Prevents Acute Doxorubicin Ovarian Insult and Follicle Demise, Improving the Fertility Window and Pup Birth Weight in Mice

<div><p>Increasing numbers of female patients survive cancer, but succumb to primary ovarian insufficiency after chemotherapy. We tested the hypothesis that Bortezomib (Bort) protects ovaries from doxorubicin (DXR) chemotherapy by treating female mice with Bort 1 hour prior to DXR. By preventing DXR accumulation in the ovary, Bort attenuated DXR-induced DNA damage in all ovarian cell types, subsequent γH2AFX phosphorylation, and resulting apoptosis in preantral follicles. Bort pretreatment extended the number of litters per mouse, improved litter size and increased pup weight following DXR treatment, thus increasing the duration of post-chemotherapy fertility and improving pup health. As a promising prophylactic ovoprotective agent, Bort does not interfere with cancer treatment, and is currently used as a chemotherapy adjuvant. Bort-based chemoprotection may preserve ovarian function in a non-invasive manner that avoids surgical ovarian preservation, thus diminishing the health complications of premature menopause following cancer treatment.</p></div

Bort pretreatment prevents DXR-induced dsDNA breaks in ovarian cells.

<p>Summary data quantify dsDNA damage as the comet moment utilizing the comet assay. Trend lines included for visualization. Panels summarize DNA damage in granulosa cells (<i>A</i>.) and stromal cells (<i>B</i>.) as time post-DXR injection plotted against comet moment. <i>C</i>. Bar graph summarizes DNA damage in oocytes 24 hrs post-DXR injection. n = 3 animals/group/time point/replicate, 4 replicates total. *p<0.05, one-way ANOVA.</p

Bort pretreatment prevented DXR accumulation in the mouse ovary.

<p><i>A</i>. Micrographs of mouse ovarian sections obtained by spectral confocal imaging (ex. 488 nm, em. 520–720 nm). Images are overlays of all collected emission wavelengths. Bar = 40 µm. To ensure control autofluorescence was visible in print, images were adjusted equally to a threshold of 140 in Photoshop with no other image enhancements. <i>B</i>. Graph plots mean fluorescence intensity +/− SEM quantified from raw DXR fluorescence in ovarian sections representing the top, middle, and bottom third of the ovary. Emission profiles at 550–560 nm (cold finger) were not collected by the microscope to prevent direct detection of the excitation laser at that wavelength. Confocal parameters were identical from one sample to the next. DXR points are statistically significant from control and Bort-DXR with p<0.05, one-way ANOVA, Bonferroni means comparison.</p

Bort pretreatment increases pup number per litter and ovarian weight following DXR.

<p>Number of pups birthed in each litter were counted and weighed for each treatment group. <i>A</i>. Plot depicts the mean number of pups per litter (± SE) for each litter number for each dam treatment group. Linear fit for Bort-DXR has a slope of 0.35±0.06 SE, DXR linear slope  = −0.31±0.04. *p = 0.033 for DXR vs. Bort-DXR, two-way ANOVA. Only 1 DXR-treated mouse achieved a sixth litter; the point is not plotted as no error can be calculated. <i>B</i>. Graph plots mean weight of ovary (mg) ± SE for each treatment group at 8 months of age [ctl = 10.4±0.4 mg (n = 36), DXR = 7.6±1.2 mg (n = 8), Bort-DXR = 9.1±0.7 mg (n = 22), Bort = 11.3±0.7 mg (n = 32)]. DXR induced a significant reduction in ovary weight (p<0.05, one-way ANOVA). Bort-DXR and Bort treatment groups were not significantly different from control.</p

Bort pretreatment improves pup weight following DXR.

<p>Pup weight, g, is plotted ± SE for each treatment group. DXR reduced pup birth weight to 86% of control, where Bort treatment groups improved pup weight to 94% of control. Asterisks denote p<0.001, one-way ANOVA with Bonferroni means comparison (n = ≥170 pups per treatment).</p

Bort pretreatment prevented DXR-induced apoptosis in mouse ovarian follicles.

<p><i>A</i>. Micrographs of mouse ovaries stained with TUNEL (green) or PI (red, nuclei), bar  = 40 µm). Representative images from 3 different mice are shown for each treatment condition. Insets are digital magnification. <i>B</i>. Bar graph quantifies the apoptotic index per follicle class calculated as fraction apoptotic/total follicles for each class. n = 4 mice *p<0.05, one-way ANOVA, Bonferroni means comparison. The very low levels of TUNEL-positive follicles in Bort-treated mice were not quantified.</p

Bort pretreatment improves the infertility index following DXR treatment.

<p>Plot represents the ‘infertility index’ plotted as the percentage of surviving animals from each treatment group that fail to achieve the next birth as a function of birth (litter) number. Symbols correspond to control, DXR, Bort –DXR and Bort treatment groups as indicated. DXR points in the linear range were statistically different from Bort-DXR, Bort, and control (p<0.05, one-way ANOVA).</p

Western blot with corresponding quantification reveals DXR-induced changes in H2AFX phosphorylation were blocked by Bort pre-treatment.

<p>Blot probed with anti-phospho H2AFX antibodies revealed an increase in density of the corresponding 17 kDa band in ovarian lysates harvested 6 hrs post-DXR injection compared to ctl, which is absent in Bort-pretreated samples. *p<0.05, one-way ANOVA, Tukey means comparison. Blot shows β-actin as the loading control. N = 3 blots/quantification.</p