Granulosa Cell Death is a Significant Contributor to DNA-Damaging Chemotherapy-Induced Ovarian Insufficiency

Abstract Typically, DNA-damaging chemotherapy (CTx) regimens have a gonadotoxic effect and cause premature ovarian insufficiency (POI), characterized by infertility and estrogen deficiency. However, whether loss of granulosa cells killed directly by CTx contributes significantly to POI has not been determined. To address this issue, we used a previously established mouse model of CTx-induced POI. The alkylating drugs Busulfan (8.75 mg/kg) and Cyclophosphamide (100 mg/kg) were administered to 8-week-old FVB female mice by intraperitoneal (i.p.) injection three times at 48-hour intervals, after which ovarian tissues were harvested and examined by immunofluorescence. The number of primordial follicles was significantly reduced at day (d)6, whereas the number of growing follicles was relatively unchanged. CTx led to DNA double strand breaks in both oocytes and granulosa cells based on the presence of γH2AX foci. However, markers of apoptosis predominantly labeled granulosa cells in growing follicles. We next examined the effect of inhibiting apoptosis in growing granulosa cells by generating Bak−/-Baxfxfx; Cyp19a1Cre transgenic mice. On d10 after the first CTx, Bak−/-Baxfxfx; Cyp19a1Cre ovaries had fewer apoptotic granulosa cells and more surviving follicles than controls. Furthermore, Bak−/-Baxfxfx; Cyp19a1Cre mice showed better fertility than controls after CTx. Our data suggest that granulosa cell death is a significant contributor to follicle depletion and fertility loss after Cyclophosphamide and Busulfan.