title: Irinotecan metabolite SN38 results in germ cell loss in the testis but not in the ovary of prepubertal mice.
abstract: Does the Irinotecan metabolite 7-ethyl-10-hydroxycamptothecan (SN38) damage the gonads of male and female prepubertal mice?
  
  
  
  The Irinotecan metabolite SN38 reduces germ cell numbers within the seminiferous tubules of mouse testes at concentrations that are relevant to cancer patients, while in contrast it has little if any effect on the female germ cell population.
  
  
  
  Little is known about the role of the chemotherapeutic agent Irinotecan on female fertility, with only one article to date reporting menopausal symptoms in perimenopausal women treated with Irinotecan, while no data are available either on adult male fertility or on the impact of Irinotecan on the subsequent fertility of prepubertal cancer patients, female or male.
  
  
  
  Male and female gonads were obtained from postnatal day 5 C57BL/6 mice and exposed in vitro to a range of concentrations of the Irinotecan metabolite SN38: 0.002, 0.01, 0.05, 0.1 or 1 µg ml-1for the testis and 0.1, 1, 2.5 or 5 µg ml-1for the ovary, with treated gonads compared to control gonads not exposed to SN38. SN38 was dissolved in 0.5% dimethyl sulfoxide, with controls exposed to the same concentration of diluent. The number of testis fragments used for each analysis ranged between 3 and 9 per treatment group, while the number of ovaries used for each analysis ranged between 4 and 12 per treatment group.
  
  
  
  Neonatal mouse gonads were developed in vitro, with tissue analysed at the end of the 4-6 day culture period, following immunofluorescence or hematoxylin and eosin staining. Statistical analyses were performed using one-way ANOVA followed by Bonferroni post-hoc test for normally distributed data and Kruskal-Wallis test followed by Dunns post-test for non-parametric data.
  
  
  
  Abnormal testis morphology was observed when tissues were exposed to SN38, with a smaller seminiferous tubule diameter at the highest concentration of SN38 (1 µg ml-1, p < 0.001 versus control) and increased number of Sertoli cell-only tubules at the two highest concentrations of SN38 (0.1 µg ml-1, p < 0.001; 1 µg ml-1, p < 0.0001, both versus control). Within seminiferous tubules, a dose response decrease was observed in both germ cell number (mouse vasa homologue (MVH)-positive cells) and in proliferating cell number (bromodeoxyuridine (BrdU)-positive cells), with significance reached at the two highest concentrations of SN38 (0.1 µg ml-1, p < 0.01 for both; 1 µg ml-1, p < 0.001-MVH, p < 0.01-BrdU; all versus control). No change was seen in protein expression of the apoptotic marker cleaved caspase 3. Double immunofluorescence showed that occasional proliferating germ cells were present in treated testes, even after exposure to the highest drug concentration. When prepubertal ovaries were treated with SN38, no effect was seen on germ cell number, apoptosis or cell proliferation, even after exposure to the highest drug concentrations.
  
  
  
  As with any study using in vitro experiments with an experimental animal model, caution is required when extrapolating the present findings to humans. Differences between human and mouse spermatogonial development also need to be considered when assessing the effect of chemotherapeutic exposure. However, the prepubertal testes and ovaries used in the present studies contain germ cell populations that are representative of those found in prepubertal patients, and experimental tissues were exposed to drug concentrations within the range found in patient plasma.
  
  
  
  Our findings demonstrate that the prepubertal mouse ovary is relatively insensitive to exposure to the Irinotecan metabolite SN38, while it induces a marked dose-dependent sensitivity in the testicular germ cell population. The study identifies the importance of further investigation to identify the risk of infertility in young male cancer patients treated with Irinotecan.
  
  
  
  None.
  
  
  
  Work supported by Medical Research Grant (MRC) grant G1002118 and Children with Cancer UK grant 15-198. The authors declare that there is no conflict of interest that could prejudice the impartiality of the present research.
authors.affiliation: Centre for Integrative Physiology, University of Edinburgh, Edinburgh EH8 9XD, UK.
authors.forename: Federica
authors.initials: F
authors.lastname: Lopes
authors.affiliation: Centre for Integrative Physiology, University of Edinburgh, Edinburgh EH8 9XD, UK.
authors.forename: Rowena
authors.initials: R
authors.lastname: Smith
authors.affiliation: Centre for Integrative Physiology, University of Edinburgh, Edinburgh EH8 9XD, UK.
authors.forename: Sophie
authors.initials: S
authors.lastname: Nash
authors.affiliation: MRC Centre for Reproductive Health, University of Edinburgh, Edinburgh EH16 4TJ, UK.
authors.forename: Rod T
authors.initials: RT
authors.lastname: Mitchell
authors.affiliation: Centre for Integrative Physiology, University of Edinburgh, Edinburgh EH8 9XD, UK norah.spears@ed.ac.uk.
authors.forename: Norah
authors.initials: N
authors.lastname: Spears
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D049109:Cell Proliferation / Q000187:drug effects; D005260:Female; D005455:Fluorescent Antibody Technique; D005854:Germ Cells / Q000187:drug effects*; D066298:In Vitro Techniques; D000077146:Irinotecan; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009865:Oocytes / Q000187:drug effects; D009924:Organ Culture Techniques; D010053:Ovary / Q000166:cytology*; D013093:Spermatogonia / Q000187:drug effects; D013737:Testis / Q000166:cytology*
chemical_list: D000077146:Irinotecan; D002166:Camptothecin
keywords: apoptosis; cell proliferation; chemotherapy; fertility preservation; gonadotoxicity; oocyte; ovary; spermatogonia; testis; tissue culture