Nguyen Q N, Zerafa N, Liew S H, Findlay J K, Hickey M, Hutt K J
Department of Obstetrics and Gynaecology, The University of Melbourne, Parkville, 3052, Australia.
Development and Stem Cells Program, Monash Biomedicine Discovery Institute, and Department of Anatomy and Developmental Biology, Monash University, Clayton, 3800, Australia.
Mol Hum Reprod. 2019 Aug 1;25(8):433-444. doi: 10.1093/molehr/gaz020.
It is well established that DNA-damaging chemotherapies can cause infertility and ovarian endocrine failure by depleting the ovarian reserve of primordial follicles. Currently, no effective pharmacological therapies exist for the preservation of long-term fertility and ovarian function in female cancer patients, due to a limited understanding of the mechanisms of chemotherapy-induced follicle depletion. This study investigated the cellular targets, molecular mechanisms, and temporal course of ovarian reserve depletion following treatment with commonly used chemotherapeutic drugs. Adult female C57BL/6 mice were injected i.p. with saline, cisplatin (5mg/kg), or cyclophosphamide (300mg/kg); ovaries were harvested after 8 or 24 hours. Follicle counts showed depletion of all follicular stages 24 hours after administration of cisplatin or cyclophosphamide. Eight hours post-treatment, H2A histone family member X (γH2AX) immunofluorescence showed DNA double-stranded breaks at all follicular stages, including within primordial follicle oocytes. This staining was resolving by 24 hours, indicating that primordial follicle oocytes begin to undergo either apoptosis or repair in this timeframe. γH2AX-positive follicles were further examined to identify the specific cell types damaged. In primordial, transitional, and primary follicles, only oocytes sustained DNA damage, whereas in secondary and antral follicles, only somatic cells were affected. TUNEL staining confirmed that apoptosis occurs in these targeted cell types. Whilst multi-drug and multi-dose regimens were not examined, this study conclusively shows that cyclophosphamide and cisplatin cause direct damage to primordial follicle oocytes, which then undergo apoptosis. Therefore, future pharmacological strategies to prevent chemotherapy-induced infertility in females must specifically prevent primordial follicle oocyte death.
众所周知，DNA损伤性化疗可通过耗尽原始卵泡的卵巢储备而导致不孕和卵巢内分泌功能衰竭。目前，由于对化疗诱导卵泡耗竭机制的了解有限，尚无有效的药物疗法来保留女性癌症患者的长期生育能力和卵巢功能。本研究调查了常用化疗药物治疗后卵巢储备耗竭的细胞靶点、分子机制和时间进程。成年雌性C57BL/6小鼠腹腔注射生理盐水、顺铂（5mg/kg）或环磷酰胺（300mg/kg）；8小时或24小时后采集卵巢。卵泡计数显示，给予顺铂或环磷酰胺24小时后，所有卵泡阶段均出现耗竭。治疗后8小时，H2A组蛋白家族成员X（γH2AX）免疫荧光显示所有卵泡阶段均有DNA双链断裂，包括原始卵泡卵母细胞内。这种染色在24小时时消退，表明原始卵泡卵母细胞在这个时间段开始发生凋亡或修复。对γH2AX阳性卵泡进行进一步检查，以确定受损的特定细胞类型。在原始卵泡、过渡卵泡和初级卵泡中，只有卵母细胞遭受DNA损伤，而在次级卵泡和窦状卵泡中，只有体细胞受到影响。TUNEL染色证实这些靶向细胞类型发生了凋亡。虽然未研究多药和多剂量方案，但本研究确凿地表明，环磷酰胺和顺铂会对原始卵泡卵母细胞造成直接损伤，随后这些细胞发生凋亡。因此，未来预防女性化疗所致不孕的药理学策略必须专门防止原始卵泡卵母细胞死亡。
