A Mouse Model and Mechanism Study of Premature Ovarian Insufficiency Induced by Different Concentrations of Cyclophosphamide

doi:10.12300/j.issn.1674-5817.2024.194

Abstract

Abstract:

Objective To observe and compare the effects of different concentrations of cyclophosphamide (CTX) in inducing premature ovarian insufficiency (POI) model in mice and investigate the mechanism of injury. Methods Thirty-two 6~8-week-old female C57BL/6J mice were randomly divided into four groups (n=8 per group) using a weight-based block randomization method. The POI model was established via a single intraperitoneal injection of 75 mg/kg cyclophosphamide (CTX), 120 mg/kg CTX, 120 mg/kg CTX + 12 mg/kg Busulfan, or an equivalent volume of normal saline (control). Ovarian coefficients, serum estradiol (E₂) and follicle-stimulating hormone (FSH) levels were measured. Western blotting was performed to assess changes in ovarian expression levels of NAD-dependent deacetylase sirtuin-5 (SIRT5) and forkhead box O3a (FOXO3a) under different modeling conditions. After determining the optimal CTX concentration for modeling, an additional forty 6~8-week-old femal C57BL/6J mice were randomly divided into five groups (n=8 per group) using a weight-based block randomization method: saline control, 120 mg/kg CTX sampling at 1, 2, 7, or 14 days after modeling. Western blotting was used to evaluate temporal changes of ovarian SIRT5 and FOXO3a protein expression. Results Compared with the saline control, all concentrations of CTX (75 mg/kg CTX, 120 mg/kg CTX) and 120 mg/kg CTX + 12 mg/kg Busulfan induced POI injury in mice. The 120 mg/kg CTX group exhibited smaller changes in ovarian coefficients (P<0.001) and E₂ levels (P<0.05), whereas the 120 mg/kg CTX + 12 mg/kg Busulfan group showed rough and reduced luster fur, sluggish response and was in the worst state. Compared with the saline control group, FOXO3a expression was significantly down-regulated (P<0.05), while SIRT5 remained unchanged in the 75 mg/kg CTX group (P>0.05). In contrast, both SIRT5 (P<0.05) and FOXO3a (P<0.05) were significantly down-regulated in the 120 mg/kg CTX group. Further analysis revealed that on day 2 and 7 after 120 mg/kg CTX modeling, the expressions of SIRT5 (P<0.01) and FOXO3a (P<0.001) were significantly down-regulated, with the largest decrease observed on day 7 (SIRT5, P<0.000 1; FOXO3a, P<0.000 1). Conclusion Ovarian injury in the POI model induced by 120 mg/kg CTX is milder than that in the POI model induced by 75 mg/kg CTX. Moreover, the expression changes of SIRT5 and FOXO3a are most significant on day 7 after modeling induced by 120 mg/kg CTX, which may be related to the inhibition of the SIRT5-FOXO3a signaling pathway.

Key words: Cyclophosphamide, Premature ovarian insufficiency, Mouse model, SIRT5-FOXO3a pathway

CLC Number:

Q95-33

GONG Leilei,WANG Xiaoxia,FENG Xuewei,et al. A Mouse Model and Mechanism Study of Premature Ovarian Insufficiency Induced by Different Concentrations of Cyclophosphamide[J]. Laboratory Animal and Comparative Medicine, 2025, 45(4): 403-410. DOI: 10.12300/j.issn.1674-5817.2024.194.

Figures/Tables 3

Figure 1 Ovarian coefficients and changes of serum hormone levels in mice on day 7 after modeling with CTX or CTX+BusulfanNote： A， Ovarian index of the first batch of mice； B， Serum E2 levels in the first batch of mice； C， Serum FSH levels in the first batch of mice. Ctl represents control group； 75 CTX， 120 CTX， 120 CTX+12 Busulfan represents premature ovarian insufficiency model group established by intraperitoneal injection of 75 mg/kg cyclophosphamide （CTX）， 120 mg/kg CTX， 120 mg/kg CTX+12 mg/kg Busulfan respectively； E2 represents estradiol； FSH represents follicle-stimulating hormone. Compared with Ctl （n=8）， ?P<0.05， ??P<0.01， ???P<0.001.

Figure 2 Expressions of SIRT5 and FOXO3a in ovarian tissues on day 7 after modeling with different concentrations of CTX and CTX+BusulfanNote： A， Western blot results of SIRT5 and FOXO3a proteins on day 7 after modeling with different concentrations of CTX and CTX+Busulfan； B， The expression of SIRT5 on day 7 after modeling with different concentrations of CTX and CTX + Busulfan； C， The expression of FOXO3a on day 7 after modeling with different concentrations of CTX and CTX + Busulfan. Compared with Ctl （n=8）， ?P<0.05， ??P<0.01.

Figure 3 Expression of SIRT5 and FOXO3a in mouse ovarian tissue at different times after 120 mg/kg CTX modelingNote： A and E， The expression of FOXO3a and SIRT5 on day 1 after modeling； B and F， The expression of FOXO3a and SIRT5 on day 2 after modeling； C and G， The expression of FOXO3a and SIRT5 on day 7 after modeling； D and H， The expression of FOXO3a and SIRT5 on day 14 after modeling； I， Western blot results of FOXO3a and SIRT5 on day 1 after modeling； J， Western blot results of FOXO3a and SIRT5 on day 2 after modeling； K， Western blot results of FOXO3a and SIRT5 on day 7 after modeling； L， Western blot results of FOXO3a and SIRT5 on day 14 after modeling. Compared with Ctl （n=5）， ??P<0.01， ???P<0.001， ????P<0.000 1.

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