Comparative Analysis of Sex Differences Between Cyclophosphamide- and Paclitaxel-Induced Mouse Models of Chemotherapy-Induced Alopecia

doi:10.12300/j.issn.1674-5817.2026.094

Abstract

Abstract:

Objective To evaluate the effect of sex on phenotypic stability in mouse models of chemotherapy-induced alopecia (CIA) induced by cyclophosphamide (CYP) or paclitaxel (PTX), and to explore potential mechanisms underlying phenotypic heterogeneity in female mice. Methods Twenty-eight wild-type specific pathogen-free (SPF) C57BL/6J mice (half male and half female) aged 6-8 weeks were selected for synchronous back hair removal. On the 9th day after hair removal (T0), the mice were randomly divided into a wild-type female CYP group, a wild-type male CYP group, a female negative control group and a male negative control group. Mice in the CYP groups were intraperitoneally injected with a single dose of 120 mg/kg CYP, and mice in the negative control groups were intraperitoneally injected with the same volume of normal saline. Subsequently, the phenotypic changes of the CIA model induced by CYP were continuously observed and photographed. Eleven 6-week-old SPF female C57BL/6J mice underwent bilateral ovariectomy (OVX group). Following successful surgery, these mice and five XY (Sry⁻) mice bred from Four Core Genotypes breeding stock were used to establish the chemotherapy-induced alopecia (CIA) model using the same protocol. Serum was collected from mice in the OVX group, wild-type female CYP group, and wild-type male CYP group on T7 after CYP administration for steroid hormone analysis by mass spectrometry to evaluate the effects of gonadal estrogen and sex chromosome background changes on the stability of the CYP-induced female alopecic phenotype. Finally, twenty SPF C57BL/6J mice aged 6-8 weeks (half male and half female) were randomly divided into a PTX low-dose group and a PTX high-dose group. On the 9th day after simultaneous hair removal, PTX was injected intraperitoneally at a single dose of 80 mg/kg or 120 mg/kg, respectively. Mice from the PTX low-dose group and the PTX high-dose group were continuously observed and photographed to compare the intra-group consistency of the phenotypes of female and male mice. Six SPF C57BL/6J mice (half male and half female) aged 6-8 weeks were used to repeat the construction method of the PTX low-dose group, and the hair follicle histological examination was performed at T0, T4, T7, T14 and T21, respectively, to observe the damage caused by PTX to hair follicle regeneration. Results The phenotypes of CYP-induced male CIA mice are stable and reproducible. There are obvious individual differences in the alopecia phenotype in the CYP-induced female CIA model mice, which may be mainly caused by the differences in the relevant estrogens in the estrous cycle. The difference between OVX+CYP group and XY (Sry⁻) +CYP group disappeared, suggesting that ovarian-derived hormones and XY (Sry⁻) sex chromosome background may be involved in the formation of phenotypic heterogeneity of CYP-induced alopecia in female mice. The mice in the high-dose PTX group and the low-dose PTX group showed a relatively consistent alopecia and regrowth process, and there was no significant sex difference. Conclusion CYP-induced CIA is strongly influenced by sex, and ovariectomy or alteration of the sex chromosome background can improve the consistency of CYP-induced alopecic phenotypes in female mice. PTX-induced CIA shows good phenotypic consistency in both female and male mice, indicating broad applicability across sexes. Taken together, the choice of animal sex in CIA model construction, mechanistic studies, and evaluation of anti-alopecia interventions should take into account the type of chemotherapeutic agent, the study objective, and the required degree of phenotypic stability. Sex should therefore be considered an important variable in model standardization and interpretation of experimental findings.

Key words: Chemotherapy-induced alopecia, Cyclophosphamide, Paclitaxel, Sex differences, C57BL/6J mice, XY（Sry ?） mice

CLC Number:

ZHONG Qiuming,QIAN Liping,CAI Yuanyuan,et al. Comparative Analysis of Sex Differences Between Cyclophosphamide- and Paclitaxel-Induced Mouse Models of Chemotherapy-Induced Alopecia[J]. Laboratory Animal and Comparative Medicine, 2026, 46(5): 677-688. DOI: 10.12300/j.issn.1674-5817.2026.094.

Figures/Tables 7

Figure 1 Construction workflow and observation time points of the chemotherapy-induced alopecia mouse model

Figure 2 Observations of phenotypic differences between females and males in a cyclophosphamide-induced mouse model of chemotherapy-induced alopecia

Note： On Day 0， perform simultaneous back hair removal. On day 9 after simultaneous back hair removal， the wild-type male cyclophosphamide （CYP） group （n=5） and the wild-type female CYP group （n=5） mice were each given a single intraperitoneal injection of 120 mg/kg CYP， while the control group （n=6） mice were given the same volume of normal saline intraperitoneally. At time points of Day 0 （T0）， Day 4 （T4）， Day 7 （T7）， Day 14 （T14）， and Day 21 （T21） after administration， observe the back hair condition of each group of mice. Red boxes indicate representative examples of inconspicuous alopecia.

Figure 3 Alopecia score results of wild-type female cyclophosphamide-treated and wild-type male cyclophosphamide-treated group mice

Note： On day 9 after synchronized dorsal depilation， mice in the wild-type male and female cyclophosphamide （CYP） groups received a single intraperitoneal injection of CYP （120 mg/kg）. T7 and T14 were days 7 and 14 after CYP intraperitoneal injection， respectively. Five mice of each sex were included. The same animals were evaluated at both T7 and T14， and each dot represents the score of an individual mouse.

Figure 4 Steroid hormone levels in serum of wild-type female cyclophosphamide group， wild-type male cyclophosphamide group， and ovariectomized + cyclophosphamide group mice

Note： Serum levels of progesterone （A）， 17β-estradiol （B）， and testosterone （C） in the wild-type female cyclophosphamide （CYP）， wild-type male CYP， and ovariectomized CYP-treated （OVX+CYP） groups on day 7 （T7） after intraperitoneal administration of cyclophosphamide （120 mg/kg）. The OVX model was established by bilateral ovariectomy， followed by a 1-week recovery period. OVX and wild-type mice then underwent synchronized dorsal depilation. On day 9 after depilation （T0）， mice in the wild-type male CYP （n=6）， wild-type female CYP （n=6）， and OVX+CYP （n=6） groups received a single intraperitoneal injection of CYP （120 mg/kg）. Each dot represents an individual mouse； ^ns P>0.05， ^* P<0.05， ^*** P<0.001.

Figure 5 Ovariectomy and deletion of the testis determining gene <i>Sry</i> <sup>-</sup> can improve the phenotypic consistency of the cyclophosphamide-induced mouse model of chemotherapy-induced alopecia

Note： Changes in dorsal hair coverage in the OVX+cyclophosphamide （CYP） and XY （Sry⁻）+CYP groups at different observation time points. Mice in the OVX+CYP group underwent bilateral ovariectomy followed by a one-week recovery period. Mice in both the OVX+CYP and XY （Sry⁻）+CYP groups then underwent synchronized dorsal depilation. On day 9 after depilation （T0）， mice in the OVX+CYP group （n=5） and XY （Sry⁻）+CYP group （n=5） received a single intraperitoneal injection of CYP （120 mg/kg）. Day 0 and Day 7 were the 0 and 7 days after simultaneous hair removal. T0， T4， T7， T14， and T21 correspond to days 0， 4， 7， 14， and 21 after CYP administration， respectively.

Figure 6 Phenotypic differences between female and male mice in a paclitaxel-induced chemotherapy alopecia mouse model

Note： Changes in dorsal hair coverage in the low-dose and high-dose paclitaxel （PTX） groups at different time points after intraperitoneal PTX administration. On day 9 （T0） after synchronized dorsal depilation， mice in the low-dose PTX group （80 mg/kg， n=10） and high-dose PTX group （120 mg/kg， n=10） received a single intraperitoneal injection of PTX at the respective doses. Day 0 and Day 7 were the 0 and 7 days after simultaneous hair removal， respectively. T0， T4， T7， T14 and T21 were days 0， 4， 7， 14 and 21 after PTX administration， respectively. For the phenotypic assessments shown in this figure， both the low- and high-dose PTX groups included five male and five female mice.

Figure 7 HE staining of hair follicle tissues in the low-dose paclitaxel-induced chemotherapy alopecia mouse model

Note： Six SPF C57BL/6J mice （half male and half female） were given a single intraperitoneal injection of 80 mg/kg PTX on the 9th day after synchronous back hair removal， and continuously observed on days 0 （T0）， 4 （T4）， 7 （T7）， 14 （T14） and 21 （T21） after administration. Scale bar=100 μm.

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