Preparation and Evaluation of Mouse Premature Ovarian Insufficiency Model

Summary

This protocol delineates a method for constructing a premature ovarian insufficiency mouse model via cyclophosphamide injection. The model effectively, readily, and adaptably replicates the pathological progression of human premature ovarian insufficiency, demonstrating high reliability and cost-effectiveness.

Abstract

Premature ovarian insufficiency (POI) is a critical condition leading to female infertility, necessitating reliable animal models for mechanistic and therapeutic research. Here, we present a standardized protocol for establishing and evaluating a cyclophosphamide (CTX)-induced POI mouse model. Six-to-eight-week-old female mice with regular estrous cycles were selected and subjected to intraperitoneal CTX injections: an initial dose of 100 mg/kg on day 1, followed by daily doses of 20 mg/kg for the subsequent 14 days. Dynamic changes in estrous cycles were monitored via vaginal smear cytology with Wright staining. Serum levels of estradiol (E2), follicle-stimulating hormone (FSH), and anti-Müllerian hormone (AMH) were quantified using ELISA to assess endocrine alterations. Ovarian histopathology was evaluated through hematoxylin-eosin (H&E) staining of paraffin-embedded sections to quantify follicular atresia, while immunohistochemical analysis of cleaved caspase-3 was performed to detect granulosa cell apoptosis. Results demonstrated disrupted estrous cyclicity, significantly reduced E2 and AMH levels, elevated FSH concentrations, increased follicular atresia, and enhanced granulosa cell apoptosis in CTX-treated mice, confirming successful POI modeling. This model-building method can highly mimic the mechanism of chemotherapy-induced ovarian damage, presenting typical pathological features such as follicle reserve depletion and sex hormone disorders. It provides a reliable experimental platform for revealing the reproductive toxicity mechanism of chemotherapy, screening ovarian-protecting drugs, and optimizing fertility preservation strategies. Moreover, this model is relatively simple to operate, low-cost, and has a short production cycle, making it easy to carry out and popularize. The methodology aligns with the requirements of JoVE for visualizable, step-by-step experimental demonstrations.

Introduction

Premature ovarian insufficiency (POI), the most prevalent form of female reproductive aging disorder, not only severely compromises patients' physical and mental health through estrogen deficiency symptoms and reproductive dysfunction, but also elevates the risks of developing comorbidities, including fractures, autoimmune disorders, cardiovascular diseases, diabetes mellitus, etc.¹. The development of efficient and safe therapeutic strategies has thus become a critically urgent priority in modern medicine. Given the limited availability of clinical samples, multifactorial etiology, and high ethical risks associated with POI research, the establishment of reliable animal models that permit systematic mechanistic investigation and safety validation has become indispensable.

Currently, chemotherapy-induced ovarian damage models are widely utilized for POI studies. Cyclophosphamide (CTX), an alkylating agent with immunosuppressive properties, has been identified as a potent gonadotoxic compound. Its mechanism involves DNA crosslinking damage to suppress cellular proliferation², leading to apoptosis of ovarian reserve cells^3,4. CTX-based models effectively recapitulate clinical hallmarks of POI, including primordial follicle depletion, hypoestrogenemia, and elevated follicle-stimulating hormone (FSH) levels. Intraperitoneal CTX injection in mice offers distinct advantages: high bioavailability through rapid peritoneal absorption, standardized operability with minimal technical variability, and cost-effectiveness for large-scale studies. Notably, the ovarian structure of mice is highly similar to that of humans, which facilitates the dynamic observation of follicular depletion and hormonal changes. Moreover, mice are low-cost and have a fast reproductive rate, meeting the requirements for large-scale sample studies.

Despite these advantages, heterogeneity in CTX dosing regimens (e.g., single-bolus versus fractionated injections) and temporal administration windows has led to inconsistent modeling outcomes across studies. To address this gap, we present a systematic protocol optimizing CTX dosage and administration frequency for POI induction in mice. This study aims to establish a reproducible model through quantitative assessment of ovarian reserve depletion, hormonal dysregulation, and histopathological correlates, thereby providing a robust platform for mechanistic exploration and therapeutic screening.

Protocol

This protocol has been reviewed and approved by the Ethics Committee of Anhui Shendong Biotechnology Development Co., Ltd. (Ethics Number: SDLL202502281). This study adheres to the guidelines of the National Institutes of Health on the care and use of laboratory rodents in all animal experiment procedures. The female C57BL/6 mice used in this experiment were 6-8 weeks old and maintained under specific pathogen-free (SPF) conditions. As an inbred strain and a substrain of the C57 lineage, C57BL/6 mice are characterized by their black coat. Prior to experimentation, the mice were acclimatized for one week in the animal laboratory under controlled temperature and humidity conditions, with a 12-h light/dark cycle to mimic natural circadian rhythms. Throughout the acclimatization period, the mice were provided ad libitum access to food and water.

1. Animal preparation

NOTE: Select 24 female SPF-grade C57BL/6 mice aged 6-8 weeks with normal estrous cycles. Collect vaginal lavage fluid from the mice at 9:00 a.m. for 10 consecutive days to prepare smears of exfoliated vaginal cells. Observe the estrous cycles and choose the mice that have at least one continuous and complete cycle.

1. Allow mice to acclimate to the environment for 1 week prior to experiments. Mark mice using an ear punch after the adaptation period. Record body weight measurements immediately after marking.
2. Securely grip mice until natural stress-induced urination occurs. Immobilize by pinching the dorsal neck skin between the thumb and forefinger of the left hand. Position the mouse head down to fully expose the abdominal region.
3. Load a sterile pipette tip onto a micro-pipette. Aspirate 10 µL of sterile physiological saline solution. Perform three sequential flush cycles through the vaginal canal. Retain lavage fluid within the pipette tip.
4. Evenly distribute the lavage fluid across the glass microscope slide. Air-dry slides completely at room temperature. Fix cells by immersing them in 95% ethanol for preservation. Label slides comprehensively with experimental parameters, and maintain protected storage in light-proof slide boxes at 4 °C when not processing.
5. Wright's staining protocol
   1. Submerge slides in 95% ethanol (for 3-10 min). Rinse thoroughly under the distilled water stream.
   2. Transfer slides through 95% ethanol bath (1 min duration).
   3. Scrape oxidized film from the hematoxylin solution surface using clean filter paper. Immerse the slides in filtered hematoxylin (for 5 min).
   4. Rinse the slides with distilled water. Dip briefly (<1 s) in 1% HCl solution. Complete with an extended distilled water rinse.
   5. Prepare mixed stain using Orange G and EA50 solutions. Stain slides in the mixture (for 3 min).
   6. Pass the slides through three sequential 95% ethanol baths (5-s intervals).
   7. Treat with xylene in two stages (2 min each).
      NOTE: Operate within a fume hood to avoid inhalation of xylene vapor. Close the containers immediately after use.
   8. Permanently preserve specimens using neutral resin mounting medium
6. When reading the slide under the microscope, identify the cells with pale pink cytoplasm and dark purple nuclei as nucleated epithelial cells, irregular scaly non-nucleated pale pink cells as keratinized epithelial cells, and small round blue cells as white blood cells.
   NOTE: Determine the estrous cycle stage based on the following cytological characteristics: (1) Proestrus phase: Predominant nucleated epithelial cells. Appear as both individual cells and clustered sheets. Sparse leukocyte presence (<5% field of view); (2) Estrus phase: Abundant anucleated keratinized epithelial cells. Characteristics: large flattened morphology with irregular borders. Near absence of leukocytes and nucleated cells (<2% combined); (3) Metestrus phase: Decreasing keratinized cell population. Emerging leukocyte infiltration. Reappearing nucleated epithelial cells (20%-40% composition); (4) Diestrus phase: Dominant leukocytes and nucleated cells (>80%). Virtual absence of keratinized cells (<1%)⁵. Reference the standardized cytological atlas in Figure 1 for pattern verification.
7. Organize and analyze the data, and select 24 mice with normal estrous cycles.

2. Preparation of cyclophosphamide injection

NOTE: Cyclophosphamide is unstable to light and should be handled in the dark throughout the process. Cyclophosphamide is toxic, so protective clothing, double-layer nitrile gloves, and masks should be worn during the operation.

1. Add 200 mg of sterile pharmaceutical-grade cyclophosphamide powder to 10 mL of sterile saline. Mix thoroughly to obtain a 20 mg/mL cyclophosphamide solution. Reserve one portion for immediate use; prepare additional concentrations with the remainder.
2. Transfer 200 µL of 20 mg/mL stock to a 1.5 mL centrifuge tube wrapped in aluminum foil. Add 800 µL of sterile saline to reach 1 mL total volume. Vortex mix for 30 s to obtain 4 mg/mL solution. Prepare 14 identical aliquots (1 mL each). Store all aliquots at -20 °C in a vertical rack.

3. Injecting the cyclophosphamide solution

NOTE: Randomize 24 mice into three cohorts using a computerized system: (1) BLANK: No treatment (n = 8); (2) NS: Saline controls (n = 8); (3) POI: Cyclophosphamide group (n = 8). Inject the mice every morning at 9:00. For the POI group, intra-peritoneally inject 100 mg/kg of cyclophosphamide solution on the first day, and then continuously inject 20 mg/kg of cyclophosphamide solution for the next 14 days. Intra-peritoneally inject an equal volume of normal saline into the NS group, and leave the BALNK group untreated. Weigh all the mice before each injection, every day, and collect vaginal exfoliated cells for smearing after injection.

1. Place an isoflurane-soaked cotton ball with forceps into a sealable container. Position the mouse inside the container for 10 s inhalation anesthesia.
   NOTE: Isoflurane is a volatile anesthetic with certain toxicity and irritation. Operations should be carried out in a well-ventilated environment following institutionally approved protocols.
2. Calculate injection volume: Body weight (g) × 5 = µL. Disinfect the right lower quadrant with a 75% ethanol swab. Insert a 26 G needle at a 30-45° angle (bevel up orientation). Advance 5 mm through the abdominal wall until "pop" sensation. Deliver solution at a 50 µL/s rate.
3. Place residual liquids and contaminated equipment into light-proof waste containers labeled "PHOTOSENSITIVE WASTE". Discard isoflurane-soaked cotton balls in designated sealed containers affixed with clear HAZARDOUS WASTE labels.
4. Thaw frozen 4mg/mL aliquots in a 37 °C water bath (3 min). Recalculate daily dose: Body weight (g) × 5 = µL. Verify solution clarity prior to loading the syringe.

4. Serum collection

NOTE: After 15 days of cyclophosphamide solution injection, collect blood from the apex of the heart of all mice. Centrifuge to obtain the serum. Mice in the control group and the NS group should be sampled during the diestrus phase. POI group mice underwent time-matched sampling paired with control/NS group mice, confirmed to be in diestrus.

1. Anesthetizing the mouse
   1. Place the mouse into a transparent, airtight chamber containing an isoflurane-saturated cotton ball.
   2. Observe the mouse's behavior. Loss of righting reflex (failure to self-correct posture when gently turned supine) indicates adequate anesthesia induction. This process typically requires 2-3 min.
   3. Remove the mouse from the container. Fix its head with your left hand, and gently press the upper eyelid with your index finger. Hold a sterile cotton swab in your right hand, dip it into erythromycin eye ointment to collect a 1-2 mm diameter amount.
      1. Evenly apply the ointment across the eyeball surface and inner eyelid. Close the eyelid, then gently massage with your fingertip for 10 s to distribute the ointment uniformly. Wipe away excess ointment around the eye using a dry cotton swab.
2. Position the mouse in a supine posture on a foam board. Trim the chest hair using surgical scissors. Disinfect the thoracic and abdominal areas with 75% ethanol.
   1. Incise the skin and subcutaneous tissue along the midline. Cut through the intercostal muscles longitudinally along the left side of the sternum. Expose the heart.
3. Insert a syringe (with the needle bevel facing up) into the apex of the heart at an angle of about 30°. Advance the needle slowly and gently aspirate. Inject the obtained blood into a 1.5 mL centrifuge tube.
4. Put the obtained blood into a centrifuge. Set the centrifuge at 850 × g for 10 min at 4 °C. Use a pipette to take out the upper-layer serum and place it in a 0.5 mL centrifuge tube. Store it at -80 °C.

5. Detection of serum estrogen, FSH, and AMH

1. Put the frozen serum in a 4 °C refrigerator overnight to thaw it slowly.
2. Take the ELISA kit out of the refrigerator 30 min in advance to allow the reagents in it to return to room temperature.
3. According to the instructions of the kit, perform serial dilutions of the standard using the standard diluent. Generally, set multiple concentration gradients, such as 0, 10, 20, 40, 80, 160 pg/mL, etc.
4. Add the diluted standard and the serum samples to be tested to the corresponding wells of the ELISA plate, 100 µL per well. At the same time, set up blank control wells and add 100 µL of the sample diluent.
5. Put the ELISA plate in a 37 °C incubator for 1-2 h to allow the antigen and antibody to fully bind.
6. After the incubation is over, carefully aspirate the liquid in the wells. Wash the ELISA plate with the washing solution 3-5 times. After each wash, gently pat the ELISA plate dry to remove the residual washing solution.
7. Add the enzyme-labeled antibody working solution, 100 µL per well. Continue to incubate in a 37 °C incubator for 1 h.
8. Repeat the washing steps above, wash 3-5 times, and pat the ELISA plate dry.
9. Add 100 µL of substrate solution to each well. Gently mix by shaking. Incubate in the dark at 37 °C for 15-30 min until a clear blue color develops.
10. Add 100 µL of termination solution to each well to stop the reaction. Observe the color change from blue to yellow.
11. Use a microplate reader to read the absorbance values (A values) of each well at a wavelength of 450 nm and record the data.
12. Take the concentration of the standard as the abscissa and the corresponding absorbance value as the ordinate to draw a standard curve. Generally, use polynomial fitting to ensure that the correlation coefficient r² of the standard curve is greater than 0.99.
    1. According to the standard curve, substitute the absorbance value of the serum sample to be tested into the standard curve equation to calculate the hormone concentration in the sample.

6. Collection of ovarian tissue

1. Euthanize the still-anesthetized mouse by cervical dislocation (following institutionally approved protocols), and then use surgical scissors to open the mouse's abdomen.
2. The ovaries are located below the kidneys and are wrapped in white fat pads. Gently lift the fat pads with forceps to expose the ovaries and fallopian tubes. Use scissors to separate the ovaries from the surrounding connective tissues.
3. Place the removed ovarian tissues into 4% paraformaldehyde for fixation for 24 h.

7. Preparation of paraffin sections

1. Dehydration: Take out the ovaries and pass them through ethanol solutions of different concentrations (75%, 85%, 95%, 100%) in sequence, with each step lasting for 1 h.
2. Transparency treatment: First, treat the ovaries in a mixture of xylene and ethanol for 15 min. Then, immerse them in Xylene I and Xylene II for 15 min each.
3. Wax impregnation: Immerse the ovaries in a mixture of xylene and paraffin. Then, transfer them into Paraffin I, Paraffin II, and Paraffin III for 0.5 h each.
4. Embedding: Place the wax-impregnated ovaries in an embedding mold. Adjust the orientation of the tissues (with the cut surface facing down). Pour melted paraffin into the mold and let it cool and solidify.
5. Sectioning: Cut the paraffin block into 5-µm-thick sections using a microtome. Perform serial sectioning and gently transfer the ribbon with a brush, ensuring it forms a uniform, translucent band that lies flat without distortion when floated on a water bath

8. H&E staining

1. Dewaxing: Immerse the paraffin-embedded sections in Xylene I for 10 min. Then transfer the sections to Xylene II and soak for another 10 min.
2. Rehydration: Pass the sections through a series of ethanol solutions of different concentrations in sequence: 100% ethanol, 95% ethanol, 85% ethanol, and 75% ethanol, with an immersion time of 5 min for each. Finally, rinse the sections with distilled water for 1 min.
3. Hematoxylin staining: Submerge the sections in hematoxylin staining solution for 5 min. Rinse the sections under running water for 30 s.
4. Differentiation: Immerse the sections in 1% hydrochloric acid-ethanol solution for 1-3 s. Rinse the sections under running water for 1-2 min.
5. Cytoplasmic staining: Immerse in eosin Y solution (1 min).Blot excess stain with filter paper.
6. Dehydration: Pass the sections through a series of ethanol solutions: 85% ethanol, 95% ethanol, and 100% ethanol (twice), with an immersion time of 5 s for each.
7. Clearing: Immerse the sections in Xylene I for 5 min. Then transfer the sections to Xylene II and soak for another 5 min.
8. Mounting: Drop neutral resin onto the stained section. Carefully place a coverslip over the section.
9. Follicle counting: Observe the stained sections under a microscope. Count and record the number of follicles at different developmental stages.

9. Immunohistochemistry for Caspase-3 in ovarian tissue

1. Antigen retrieval: Place the dewaxed and rehydrated sections into sodium citrate buffer (pH 6.0). Heat the sections in a microwave oven on high power for 5 min. Then take them out and let them cool.
   1. Repeat the heating process 2 more times, for a total of 3 heating cycles. After natural cooling, wash the sections with PBS 3 times, each time for 5 min.
2. Elimination of endogenous peroxidase: Add 3% H₂O₂ drop-wise onto the sections. Incubate the sections at room temperature in the dark for 10 min. Wash the sections with PBS 3 times, each time for 3 min.
3. Serum blocking and antibody incubation: Add rabbit anti-caspase-3 primary antibody drop-wise onto the sections. Incubate the sections in a wet box at 4 °C overnight. The next day, allow the sections to return to room temperature for 30 min. Wash the sections with PBS 3 times, each time for 5 min.
4. Color development, counterstaining, and mounting: Use DAB for color development, which usually takes about 1-5 min. Counterstain the sections with hematoxylin for 2 min. After counterstaining, wash the sections for 5 min.
   1. Then place the sections in ammonia water for 1 min to turn them blue. Finally, perform dehydration through a gradient of ethanol, clear the sections, and mount them.

Results

The process of establishing the POI mouse model is illustrated in Figure 2. The body weight and estrous cycle of the mice were recorded starting from the first day of drug injection. After 15 days of CTX injection, the average body weight decline rate of the mice in the POI group (3.717% ± 1.463%) was significantly higher than that in the BLANK group (0.2526% ± 0.1469%) and the NS group (0.4305% ± 0.2494%) (p < 0.0001). There was no statistically significant difference in body we...

Discussion

Premature ovarian insufficiency (POI) seriously affects women's physical health and quality of life, and its global prevalence is increasing year by year. However, at present, there is still a lack of safe and effective treatment methods for POI. Establishing a reliable and reproducible animal model is crucial for advancing research on POI. Clinically, the causes of POI are diverse, and chemotherapy drugs are important known pathogenic factors⁸. As an alkylating agent chemotherapy drug, CTX mainly...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4% paraformaldehyde	Sangon Bioengineering Co., Ltd	A500684-0500
75% alcohol	Shandong Dexinkang Medical Technology Co., Ltd	200603
Anhydrous ethanol	Bengbu Chemical Reagent Factory
Centrifuge tubes	biosharp	BS-15-M
Cold table	Quanlin Medical	QL-LT20230003
Cotton balls	Shandong Brilliance Co., Ltd	20241008
Coverslips	Jiangsu Shitai Experimental Equipment Co., Ltd	80312-3412
Cyclophosphamide	Baxter Oncology GmbH	3K645A
Dehydrator	Hubei Xiaogan Haikuo Medical Technology Co., Ltd	KH-TS
Dyeing machine	Hubei Xiaogan Haikuo Medical Technology Co., Ltd	KH-S101
EA50 staining solution	Hubei Taikang Medical Equipment Co., Ltd	J7EC20
Ear tag pliers	Shenzhen Ruiwode Life Science and Technology	C22002-10
Efficient sectioning of paraffin	Tongxiang Hualing Wax Industry Co., Ltd	20241018
Electric blast drying oven (oven)	Shanghai Yuejin Medical Equipment Co., Ltd	HCZF-11
Electrically heated thermostatic water incubator	Shanghai Yuejin Medical Equipment Co., Ltd	HSW-600
Embedding machine	Prisstar Changzhou Medical Device Co., Ltd	PMB-A
Eosin stain	Sewell Biotechnology Co., Ltd	G1005-2
Hematoxylin differentiation solution	Sewell Biotechnology Co., Ltd	G1039-500ML
Hematoxylin staining solution	Sewell Biotechnology Co., Ltd	G1005-1
Isoflurane	Shandong Ante Animal Husbandry Technology Co., Ltd	2024101501
microscope	Anhui Jiashang Biotechnology Co., Ltd	SQS-12P
Mouse anti-Mullerian hormone (AMH) ELISA kit	Hengyuan Biotechnology	HB1334-Mu
Mouse estradiol (E2) enzyme-linked immunoassay kit	Hengyuan Biotechnology	HB975-Mu
Mouse follicle-stimulating hormone (FSH) ELISA kit	Hengyuan Biotechnology	HB967-Mu
Neutral gum	Nanchang Yulu Experimental Equipment Co., Ltd	240504
Pathological tissue bleaching apparatus	Prisstar Changzhou Medical Device Co., Ltd	PHY-III
PBS buffer	biosharp	BL302A
Pipette tips	biosharp	BS-10-T
Pipettes	Dalong Medical Equipment Co., Ltd	YE213AT0257922
Rapid tissue dehydrator	Hubei Xiaogan Haikuo Medical Technology Co., Ltd	KH-TS
slicer	Gheddy	YD-315
Slides	Jiangsu Shitai Experimental Equipment Co., Ltd	80312-3161
Smear machine	Hubei Taikang Medical Equipment Co., Ltd	131001305
Sodium chloride injection	Wuhan Binhu Shuanghe Pharmaceutical Co., Ltd	240525K04
syringe	Jiangsu Zhiyu Medical Device Co., Ltd
Vibrating slicer	Shanghai Zhixin Instrument Co., Ltd	ZQP-86
Xylene	Tianjin Kaitong Chemical Reagent Co., Ltd	20240102