A Modified Technique for Inducing Polycystic Ovary Syndrome in Mice

Summary

This protocol introduces a technique for inducing polycystic ovary syndrome (PCOS) models in mice through controlled letrozole release using mini-pumps. Under adequate anesthesia, the mini-pump was implanted subcutaneously, and PCOS was successfully induced in the mice after a certain period of the mini-pump release.

Abstract

Polycystic ovary syndrome (PCOS) is one of the leading causes of infertility in women. Animal models are widely used to study the etiologic mechanisms of PCOS and for related drug development. Letrozole-induced mouse models replicate the metabolic and reproductive phenotypes of patients with PCOS. The traditional method of letrozole treatment in PCOS mice requires daily dosing over a certain period, which can be labor-intensive and cause significant stress to the mice. This study describes a simple and effective method for inducing PCOS in mice by implanting a controlled letrozole-releasing mini-pump. A mini-pump capable of stable, continuous release of a quantitative amount of letrozole was fabricated and implanted subcutaneously in mice under anesthesia. This study demonstrated that the mouse model successfully mimicked PCOS features after letrozole mini-pump implantation. The materials and equipment used in this study are readily available to most laboratories, requiring no special customization. Collectively, this article provides a unique, easy-to-perform method for inducing PCOS in mice.

Introduction

Polycystic ovary syndrome (PCOS) is one of the most common conditions among women of reproductive age¹. It affects up to 18% of women globally and is the leading cause of female infertility worldwide^2,3. PCOS is characterized by a series of interrelated reproductive abnormalities, including disturbed gonadotropin secretion, chronic anovulation, increased androgen production, and polycystic ovarian morphology⁴. In addition to gynecological disorders, PCOS also increases the risk of cardiovascular diseases^5,6. Despite decades of research, the etiology of PCOS remains unclear^7,8.

To gain better insights into the pathogenesis of PCOS and develop novel therapies, the creation of animal models that closely mimic human physiology is of tremendous importance⁹. Currently reported rodent models of PCOS include those induced by treatments with testosterone, letrozole, and estradiol valerate, among others. Testosterone induces hyperandrogenemia and is more commonly used as a PCOS inducer in rats¹⁰. DHEA has been used to induce PCOS in rodents, increasing testosterone levels, LH/FSH (luteinizing hormone/follicle-stimulating hormone) ratios, and causing irregular estrous cycles¹¹. Estradiol valerate (EV) is a long-acting estrogen, and studies using this method show that levels of sex steroid hormones and gonadotropins vary depending on the dose of EV administered^12,13,14. Letrozole is a nonsteroidal aromatase inhibitor¹⁵. Letrozole treatment induces non-cyclic estrus, increases ovarian weight, body weight, enlarges adipocytes, maximizes follicle development, and elevates testosterone levels in rats^16,17. Letrozole-treated mice exhibit increased numbers of sinus follicles and hemorrhagic cysts, as well as elevated concentrations of LH, FSH, estradiol, and progesterone^18,19.

Currently, the main methods for letrozole-induced PCOS modeling involve oral administration and subcutaneous injection, both of which require repeated daily dosing^20,21,22. These methods are time-consuming and labor-intensive, and the repetitive administration likely causes significant stress to the animals²³. Although some studies use letrozole pellets^24,25, these products need to be customized and are expensive. This report describes a technique for inducing PCOS in mice using mini-pumps. This method is simple, time-saving, and uses surgical tools and equipment that are readily available in most laboratories.

Protocol

All animal experimental protocols in this study were approved by Animal Ethics Committee of Fudan University. Female C57BL/6J mice, aged 4 weeks, were used here. The details of the animals, reagents, and equipment used in this study are listed in the Table of Materials.

1. Preparation of the mini-pump

1. Take the letrozole powder (50 mg) stored in the original packaging bottle, centrifuge the letrozole powder in the bottle at 300 x g for 5 s (at room temperature), then open the cap.
2. Slowly add 625 µL of DMSO to the letrozole powder with a pipette and mix well to obtain a stock solution at a concentration of 8 mg/100 µL, totaling 625 µL.
   NOTE: If using a different package size of letrozole powder, please calculate the required volume of DMSO in advance to configure the stock solution. The letrozole powder, DMSO, and other related compounds must be of sterile grade. The pipette tips, tubes, and other related items should be sterile or sterilized before use.
3. Count the number of required mice according to the experimental design.
4. Draw up the total amount of letrozole stock solution required with a pipette, depending on the number of required mice, and inject it into the centrifuge tube slowly.
   NOTE: 4-week-old mice for an 8-week exposure were selected to induce the PCOS mice model. The total dose of letrozole is 8 mg for each mouse, which requires two consecutive pumps to be implanted (one pump releases for 4 weeks), i.e., 4 mg/4 weeks per pump. As mentioned in step 1.2, the concentration of the letrozole stock solution is 8 mg/100 µL. To prepare the pumps, 50 µL of stock solution is required for each pump as calculated below: a total of 4 mg letrozole divided by 8 mg/100 µL.
5. Draw up an equal amount of PEG300, add it to the letrozole solution, and mix well (it can be vortexed appropriately).
6. Prepare the control vehicle solution for the control group (50 µL of DMSO + 50 µL of PEG300 for each pump without letrozole).
7. Prepare an adequate number of sterile centrifuge tubes according to the number of pumps required; one 15 mL centrifuge tube needs to be prepared for each pump.
8. Draw saline with a syringe and add 5 mL of saline to each centrifuge tube. Then, cap the centrifuge tubes and place them on a tube rack for further use.
9. Wear a new pair of sterile gloves, take out the mini-pumps in the desired quantity, and place them on a clean, sterile countertop or container.
10. Gently pinch the cap that matches the pump body with the thumb and forefinger, and hold the pump body with the other hand.
11. Slowly poke the filling port of the pump several times with the thin tube attached to the cap (Figure 1B).
    NOTE: If not unclogged, fluid may not flow freely in the tube after filling.
12. Take the needle that matches the pump and a 1 mL sterile syringe. Attach the needle to the syringe and make it tight (Figure 1C).
13. Carefully and slowly draw up the final solution of letrozole configured above with the newly assembled syringe (Figure 1D).
    NOTE: Aspiration of the letrozole solution needs to be done slowly and evenly. Rapid aspiration may generate air bubbles, which may affect the subsequent drug solution injection.
14. Inject the letrozole solution into the mini-pump.
    1. Carefully pinch a mini-pump with one hand so that it stands upright, and with the other hand, hold the aforementioned 1 mL syringe and slowly insert it into the very bottom of the pump (Figure 1E).
       NOTE: If the needle is not inserted into the bottom of the pump, the liquid in the pump can easily bubble during injection, which will affect the pump's subsequent release of the drug.
    2. Then slowly inject the configured solution; the injection can be noticed when the opening of the pump slightly bubbles.
    3. Inject more slowly when the injection volume approaches 90 µL. Once the pump is fully filled (about 100 µL), withdraw the syringe.
    4. Carefully insert the cap into the pump body and make it tight; at this point, a small amount of solution will spill out. Carefully wipe it away.
15. Take the pump loaded with the drug, with the outlet facing upwards (the end with the cap), take the previously prepared centrifuge tube, and fully immerse the pump in sterile saline (Figure 1F). Then, cap the centrifuge tube and place it on the tube rack.
    NOTE: Take care to avoid contaminating the pump throughout the process.
16. After all the mini-pumps are made, place all the centrifuge tubes in a 37 °C thermostat for 48 h.

2. Preparation for operation and anesthesia

1. Prepare and sterilize all required instruments on the day of surgery, including forceps, hemostats, surgical scissors, needle holders, and 4-0 absorbable surgical sutures.
2. Before using the isoflurane anesthesia machine (Figure 2B), carefully check whether the pipeline connection is correct and ensure the pipeline is intact and does not leak.
3. Unscrew the filling sealing cap at the anesthesia machine's evaporator, then slowly add the anesthetic.
4. Keep the anesthetic level between the upper and lower limits labeled on the anesthesia machine.
5. Convert the three-way valve switch to ensure that the airflow from the anesthesia machine evaporator is connected to the anesthesia induction box.
6. Open the air pump and the evaporator, then adjust the concentration of isoflurane for anesthesia induction (generally 3%-4%).
7. Wait for about 1 min until the anesthetic fills the induction box, then put the mouse into the induction box and wait until complete anesthesia (this process takes about 2-3 min).
8. Check that the mice are adequately anesthetized by gently tipping the box. If the mice are turned over and show no signs of returning to the recumbent position, anesthesia is induced.

3. Implantation of the letrozole mini-pump

1. Put on a surgical mask and sterile gloves.
2. Remove the pre-anesthetized mice from the induction box and apply the depilatory cream to the back of the mice, then wait for 1 min. Gently wipe off the depilatory cream and hair with wet gauze.
3. At the same time, change the three-way valve switch to ensure that the airflow from the anesthesia machine evaporator is connected to the anesthesia mask and adjust the maintenance concentration (generally 2%).
4. After dehairing, hold the mouse with the dominant hand and place its head/nose into the anesthesia mask.
5. Check the depth of anesthesia by testing the pedal withdrawal reflex.
   NOTE: All procedures involving anesthesia must be performed using gas filters fitted with activated charcoal tablets, and the procedure should be performed in a fume hood.
6. Secure the mouse in the prone position on the operating plate and apply eye lubricant to both eyes.
7. Apply povidone-iodine swabs three times to the exposed skin (Figure 2C). Then, inject meloxicam subcutaneously at a dose of 2 mg/kg. 
8. Confirm the anesthetic depth via a toe pinch again. Then, at the midline of the back of the neck, favoring the skin on the hairless side, pick up the skin with forceps and make an incision of approximately 0.5 cm laterally with a scalpel or surgical scissors (Figure 2D).
9. Then open the epidermis underneath the incision and access it with a hemostat or needle holder to free the skin, making room for a mini-pump (roughly 2 cm away from the incision) (Figure 2E).
10. Take the pump out of the saline in the centrifuge tube with sterile forceps, with the open end of the pump facing downward, and be ready to insert the pump subcutaneously in the mice (Figure 2F).
11. Clamp up one side of the skin incision with forceps and push the pump into the subcutaneous space. There should be no resistance when entering the pump. Once inside, use the fingers to slightly and gently squeeze the pump inward as far as possible (Figure 1G).
12. Suture the incision site carefully using 4-0 surgical sutures (Figure 2H).
    NOTE: Take care not to damage the subcutaneous pump body when suturing.
13. After suturing, disinfect the skin at the surgical site twice with iodophor swabs.

4. Animal recovery

1. After surgery, gently transfer the mice from the mattress used during anesthesia to a dry, clean mattress. Ensure that the mice are in a warm and quiet environment.
2. Closely monitor the conditions of the mice, such as the depth and frequency of their breaths.
3. Observe the behavior of the mice as their mobility begins to return. Finally, ensure that they can stand and walk without any obvious abnormal behaviors such as dizziness or uncoordinated movements.
4. After the mice have recovered, gently transfer them to pre-prepared cages. Place sterile jelly and food at the bottom of the cage for the mice to feed on. 
   NOTE: The stretching of the surgical site may prevent the mice from easily drinking and eating during the early postoperative period, so place jelly and food on the bottom of the cage.
5. Inject meloxicam subcutaneously at a dose of 2 mg/kg (every 24 h for the first 3 days after surgery). After 8 weeks of letrozole treatment (i.e., two consecutive mini-pump implantations), test the mice models.

Results

The experimental protocol and some critical steps are shown in Figure 1 and Figure 2. Serum testosterone levels are displayed in Figure 3A. Letrozole mini-pump-treated mice (hereafter referred to as LTZ mice) exhibited significantly elevated serum testosterone levels compared to female control mice. Meanwhile, histological analysis of ovaries showed that LTZ mice showed polycystic ovaries with a significant reduction in corpora lute...

Discussion

This report demonstrates a simple protocol for inducing PCOS in mice using easily accessible materials. The mouse PCOS model is essential for exploring PCOS mechanisms and screening drugs²⁶. Of the available methods for inducing PCOS models in mice, letrozole induction is one of the most commonly used. The use of letrozole can develop and maintain a hyperandrogenic condition by inhibiting the conversion of testosterone to estradiol or by increasing testosterone synthesis²⁷....

Materials

Name	Company	Catalog Number	Comments
Syringe	Bofeng Biotech	BD300841	1ML (sterile)
Centrifugation tube	Biological Hope	1850-K	15ML (sterile)
Pipette	Eppendorf	3123000268-A	100μL-1000μL (sterile)
Pipette	TopPette	7010101008	10μL-100μL (sterile)
Letrozole powder	Sigma	L6545-50MG	Primary acting drugs (sterile)
PEG(Poly(ethylene glycol))	Solarbio	P8250	Used for dissolution (sterile)
Sterile Physiological Saline Solution	Biosharp	BL158A	Mini-pump storage
Osmotic Pumps	ALZET	1004	Letrozole storage and sustained release (sterile)
Dimethyl sulfoxide	Biosharp	BS087	Used for dissolution (sterile)
Small Animal Anesthesia Machine	RWD	R500IP	Used for anesthesia
Isoflurane	RWD	20071302	Used for anesthesia
Hemostats	Biosharp	BS-HF-S-125	Surgical instrument
Scissors	Biosharp	BS-SOR-S-100P	Surgical instrument
Needle holder	ShangHaiJZ	J32010	Surgical instrument
Forceps	RWD	F12028	Surgical instrument
Needle and the 4-0 absorbable suture	JINGHUAN	CR413	Surgical instrument
Povidone-iodine swabs	SingleLady	GB26368-2010	Skin disinfection
Depilatory cream	ZIKER BIOTECHNOLOGY	ZK-L2701	Depilation agent for laboratory animals
Nitrile Gloves	Biosharp	BC040-L	Used for aseptic operation
Sterile gauze	ZHENDE	BA69087	Used for wiping liquids
C57BL/6J Mice	Shanghai Model Organisms Center	N/A	Age: 4 weeks
Pet Eye Lubricant	BAITE	SHF021153	Used for mouse eye lubricant
Meloxicam Injection	MEIDAJIA	R21064-21	Used for mouse analgesia (sterile)