Acute Doxorubicin Insult in the Mouse Ovary Is Cell- and Follicle-Type Dependent

Abstract

Primary ovarian insufficiency (POI) is one of the many unintended consequences of chemotherapy faced by the growing number of female cancer survivors. While ovarian repercussions of chemotherapy have long been recognized, the acute insult phase and primary sites of damage are not well-studied, hampering efforts to design effective intervention therapies to protect the ovary. Utilizing doxorubicin (DXR) as a model chemotherapy agent, we defined the acute timeline for drug accumulation, induced DNA damage, and subsequent cellular and follicular demise in the mouse ovary. DXR accumulated first in the core ovarian stroma cells, then redistributed outwards into the cortex and follicles in a time-dependent manner, without further increase in total ovarian drug levels after four hours post-injection. Consistent with early drug accumulation and intimate interactions with the blood supply, stroma cell-enriched populations exhibited an earlier DNA damage response (measurable at 2 hours) than granulosa cells (measurable at 4 hours), as quantified by the comet assay. Granulosa cell-enriched populations were more sensitive however, responding with greater levels of DNA damage. The oocyte DNA damage response was delayed, and not measurable above background until 10–12 hours post-DXR injection. By 8 hours post-DXR injection and prior to the oocyte DNA damage response, the number of primary, secondary, and antral follicles exhibiting TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)-positive granulosa cells plateaued, indicating late-stage apoptosis and suggesting damage to the oocytes is subsequent to somatic cell failure. Primordial follicles accumulate significant DXR by 4 hours post-injection, but do not exhibit TUNEL-positive granulosa cells until 48 hours post-injection, indicating delayed demise. Taken together, the data suggest effective intervention therapies designed to protect the ovary from chemotherapy accumulation and induced insult in the ovary must act almost immediately to prevent acute insult as significant damage was seen in stroma cells within the first two hours.

In situ cultured preantral follicles is a useful model to evaluate the effect of anticancer drugs on caprine folliculogenesis

“…Moreover, an apparent increase in the H2AXph139 was also observed in the presence of positive foci in the granulosa cells of the primordial follicles, oocytes from the developing follicles and in the stromal cells post treatment with 0.03µg/mL DXR. On the other hand, Roti et al, () did not observe an increase in the TUNEL and H2AXph139 positive foci in the primordial follicles after 48 hours of treatment in mice treated with 20 mg/kg of DXR. This study indicates that the DNA damage in the primordial follicles is only observed after a long exposure to DXR.…”

“…DXR acts by blocking the topoisomerase II action and inducing the production of the reactive oxygen species, which triggers cell death by blocking the protein synthesis and promoting oxidative damage (Tokarska et al, ). Studies in vivo on the DXR action (20 mg/kg) on mice ovary, showed that the accumulation of this drug in the follicles varies with the developmental stage in which the larger follicles (secondary, tertiary and antral) accumulated significantly more doxorubicin than did the smaller follicles (primordial and primary) (Roti et al, ). In the present study it was also observed that the developing preantral follicles cultured with 0.03 µg/mL DXR, had a higher number of foci positive for active caspase‐3 and H2AXph139 compared with those treated with PTX (0.1 or 0.001 µg/mL).…”

“…Recent advances in the treatment of neoplastic diseases have increased the long‐term survival of patients undergoing chemo‐radiotherapy (Roti et al, ). However, the utilization of cancer medications can lead to gonadal failure and affect all aspects of reproductive health, including prepubescent development, hormone production, and sexual functioning in adults.…”

Single and combined effects of cisplatin and doxorubicin on the human and mouse ovary in vitro

“…CIS also increases phosphorylation of components of the PTEN/Akt/FOXO3a pathway that regulates growth activation of primordial follicles (Chang et al 2015, Jang et al 2016. DOX induces DNA damage in somatic cells (Roti Roti et al 2012, Xiao et al 2017. Apoptosis and proliferation of ovarian cortical stromal tissue was affected by drug exposure.…”

Comet assay on thawed embryos: An optimized technique to evaluate DNA damage in mouse embryos

“…The exposure of parents to environmental toxicants, such as polycyclic aromatic hydrocarbons (Einaudi et al, 2014;Perrin et al, 2011), Bisphenol A (Goldstone et al, 2015), solvents (Kolstad et al, 1999), metals (Thompson and Bannigan, 2008;Zhou et al, 2016) or various therapies (Bujan et al, 2014;Esquerré-Lamare et al, 2015;Pecou et al, 2009;Roti Roti et al, 2012) may induce DNA damage in male and female germ cells. DNA damage in parental germ cells can lead to reproductive issues, such as reduced fertilization, impaired early embryonic development, a decreased pregnancy rate and increased miscarriage rate (Simon et al, 2014;Zhao et al, 2014).…”