Evidence of apoptosis as an early event leading to cyclophosphamide-induced primordial follicle depletion in a prepubertal mouse model

Front Endocrinol (Lausanne). 2024 Oct 14:15:1322592. doi: 10.3389/fendo.2024.1322592. eCollection 2024.

Abstract

Introduction: The mechanisms leading to ovarian primordial follicle depletion following gonadotoxic chemotherapy with cyclophosphamide and other cytotoxic drugs are currently understood through two main explanatory theories: apoptosis and over-activation. Discrepancies between the findings of different studies investigating these mechanisms do not allow to reach a firm conclusion. The heterogeneity of cell types in ovaries and their different degrees of sensitivity to damage, cell-cell interactions, periodical follicle profile differences, model age-dependent differences, and differences of exposure durations of tested drugs may partially explain the discrepancies among studies.

Methods: This study used intact prepubertal mice ovaries in culture as study model, in which most follicles are primordial follicles. Histological and transcriptional analyses of ovaries exposed to the active metabolite of cyclophosphamide 4-hydroperoxycyclophosphamide (4-HC) were carried out via a time-course experiment at 8, 24, 48, and 72 h.

Results: 4-HC treated ovaries showed a significant decrease in primordial follicle density at 24 h, along with active DNA damage (TUNEL) and overexpressed apoptosis signals (cleaved-poly ADP ribose polymerase in immunohistochemistry and western blotting). Meanwhile 4-HC treatment significantly up-regulated H2ax, Casp 6, Casp 8, Noxa, and Bax in ovaries, and up-regulated Puma in primordial follicles (FISH).

Discussion: Our results indicated that cyclophosphamide-induced acute ovarian primordial follicle depletion was mainly related to apoptotic pathways. No evidence of follicle activation was found, neither through changes in the expression of related genes to follicle activation nor in the density of growing follicles. Further validation at protein level in 4-HC-treated prepubertal mice ovaries at 24 h confirmed these observations.

Keywords: apoptosis; cyclophosphamide; gene expression; growing follicle; ovary; overactivation; primordial follicle; timepoint.

MeSH terms

  • Animals
  • Antineoplastic Agents, Alkylating / adverse effects
  • Antineoplastic Agents, Alkylating / pharmacology
  • Antineoplastic Agents, Alkylating / toxicity
  • Apoptosis* / drug effects
  • Cyclophosphamide* / adverse effects
  • Cyclophosphamide* / pharmacology
  • Cyclophosphamide* / toxicity
  • DNA Damage / drug effects
  • Female
  • Mice
  • Ovarian Follicle* / drug effects
  • Ovarian Follicle* / metabolism
  • Sexual Maturation / drug effects

Substances

  • Cyclophosphamide
  • Antineoplastic Agents, Alkylating

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research was funded by the Swedish Childhood Cancer Foundation (PR2016-0115, PR2020-0136), the Swedish Cancer Society (CAN 2017/704, 20 0170 F), the Swedish Research Council (Dnr 2020-02230), Radiumhemmets Research Funds Grant for clinical researchers 2020-2025, the Stockholm County Council (FoUI-953912) and the Karolinska Institutet Research grants in pediatrics from the Birgitta and Carl-Axel Rydbeck Donation, 2020-00339 to KAR-W, grants from the Shenzhen Science and Technology Program (KQTD20190929172749226 to KL), the National Natural Science Foundation of China and the Swedish Research Council Collaboration Research Programme (NSFC-VR 8211101255 to KL and KAR-W), XH has been supported by the China Scholarship Council.