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.
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.
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 worldwide2,3. 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 diseases5,6. Despite decades of research, the etiology of PCOS remains unclear7,8.
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 administered12,13,14. 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 rats16,17. Letrozole-treated mice exhibit increased numbers of sinus follicles and hemorrhagic cysts, as well as elevated concentrations of LH, FSH, estradiol, and progesterone18,19.
Currently, the main methods for letrozole-induced PCOS modeling involve oral administration and subcutaneous injection, both of which require repeated daily dosing20,21,22. These methods are time-consuming and labor-intensive, and the repetitive administration likely causes significant stress to the animals23. Although some studies use letrozole pellets24,25, 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.
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
2. Preparation for operation and anesthesia
3. Implantation of the letrozole mini-pump
4. Animal recovery
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 lutea number (Figure 3B,C), indicating anovulation. Moreover, LTZ mice exhibited significant estrous acyclicity compared to female control mice (Figure 3D). The evaluation of body weight revealed that LTZ mice were heavier than control mice (Figure 3E). Glucose tolerance test results and AUC calculations demonstrated that LTZ mice developed glucose intolerance compared to control mice (Figure 3F,G). Serum lipid assay indicated that LTZ mice exhibited a change in serum lipid profile compared to control mice (Figure 3H).
Figure 1: Materials and critical steps for preparing the mini-pump. (A) Schematic illustration showing the preparation of letrozole mini-pumps. (B) The mini-pump was unblocked by a thin tube attached to the cap. (C) A needle matching the pump was attached to the needle end of a 1 mL syringe. (D) Letrozole solution was slowly aspirated without bubbling. (E) Letrozole solution was injected slowly from the very bottom of the pump. (F) The filled pumps were capped and stored in saline with the cap end pointing upwards. Please click here to view a larger version of this figure.
Figure 2: Materials and critical steps of implanting the pumps in the mice for PCOS induction. (A) Schematic illustration showing implantation of letrozole mini-pumps. (B) Anesthesia equipment: air pump, anesthesia machine, induction box, and gas filters. (C) Anesthetized mice were fixed and sterilized. (D) A 0.5 cm cut near the dorsal midline. (E) The skin was freed to make space for mini-pump insertion. (F) A mini-pump was clipped out of the saline with clean and sterile forceps. (G) The mini-pump was implanted under the previously freed skin. (H) The skin was closed with 4-0 sutures. (I) The implanted mouse was allowed to recover. Please click here to view a larger version of this figure.
Figure 3: Characterization of reproductive and metabolic phenotypes in the letrozole mini-pump implanted mouse models. (A) Plasma testosterone levels in the LTZ-treated mice were significantly higher than those in the control mice (n = 9 animals per group). (B) Histological analysis of the ovaries.The arrow indicates corpora lutea. Scale bar: 1 mm. (C) Number of corpora lutea in the indicated groups (n = 9 animals per group). (D) LTZ mice showed acyclicity compared with control female mice. (E) The body weight in the indicated groups (n = 10 animals per group). (F,G) Glucose tolerance test results in indicated groups (n = 8 animals per group). (H) Serum lipid levels in indicated groups (n = 8 animals per group). Data are expressed as mean ± SEM. AUC indicates area under the curve; E, estrus stage; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; P, proestrus stage; M/D, metestrus/diestrus stage; TC, total cholesterol; and TG, triglycerides. Please click here to view a larger version of this figure.
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. The letrozole-induced mouse model shows metabolic and reproductive similarities to patients with PCOS28. However, conventional techniques of letrozole treatment in the mouse PCOS model require daily oral feeding or injection of letrozole drug into mice for 21 days or more29,30. Long-term continuous drug allocation with timed dosing of feeding or injection is time-consuming and labor-intensive, and repeated administration of drugs can be notoriously stressful for mice, introducing non-experimental factors affecting the model results23,31. Although some rat and mouse PCOS studies use implantable letrozole pellets28,32, the products used need to be customized and may not be available in a timely manner. The current study proposes a technique of implanting letrozole mini-pumps to induce PCOS in mice. The method is simple, time-saving, and uses surgical tools and equipment readily available in most laboratories.
Critical steps and troubleshooting
It is essential to note a few key steps to achieve optimal results when practicing this method. Firstly, unclog the pump using the thin tube attached to the matching cap before use. Previous experience indicates that a clogged pump can disrupt drug loading and release. Additionally, each step in configuring the letrozole solution must be executed with care to ensure the final solution is precisely at the desired concentration. Throughout the process of assembling the pump, attention must be paid to prevent contamination. Furthermore, careful adjustments are necessary before and during anesthesia to ensure adequate anesthesia for the experimental mice and to minimize the adverse effects of anesthesia on the operated mice.
Advantages and limitations
The method described in this study offers several advantages: (1) it saves time and allows for continuous letrozole release post-pump implantation; (2) it avoids the stress of repeated drug administration on experimental animals and minimizes the influence of non-experimental factors on modeling results; (3) the necessary materials, surgical tools, and equipment are commonly available in most laboratories; (4) the method effectively induces PCOS phenotypes in mice. However, this method also has limitations: (1) the anesthesia and surgical procedure required for mini-pump implantation may cause irritation to the mice, and the presence of the mini-pump under the skin could disturb their activity; (2) the efficacy of this modeling method may be impacted by contamination or damage to the pump body during the process. Based on the authors' experience, meticulous execution of the protocol steps can lead to a high success rate in inducing the PCOS mouse model.
The authors have nothing to disclose.
The study was supported by National Key Research and Development Program of China (grants 2021YFC2700701), the National Natural Science Foundation of China (grants 82088102, 82071731, 82171613, 8227034, 81601238), Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (grant 2019-I2M-5-064), the Science and Technology Commission of Shanghai Municipality (grants 21Y11907600), Shanghai Municipal Commission of Health and Family Planning (grant 20215Y0216), Collaborative Innovation Program of Shanghai Municipal Health Commission (grant 2020CXJQ01), Clinical Research Plan of Shanghai Hospital Development Center (grant SHDC2020CR1008A), Shanghai Clinical Research Center for Gynecological Diseases (grant 22MC1940200), Shanghai Urogenital System Diseases Research Center (grant 2022ZZ01012), Shanghai Frontiers Science Research Base of Reproduction and Development, The Science and Technology Commission of Quzhou Municipality (grant 2022K54), Open Fund Project of Key Laboratory of Reproductive Genetics, Ministry of Education, Zhejiang University (grant KY2022035), and Open Fund Project of Guangdong Academy of Medical Sciences (grant YKY-KF202202).
C57BL/6J Mice | Shanghai Model Organisms Center | N/A | Age: 4 weeks |
Centrifugation tube | Biological Hope | 1850-K | 15ML |
Depilatory cream | ZIKER BIOTECHNOLOGY | ZK-L2701 | Depilation agent for laboratory animals |
Dimethyl sulfoxide | Biosharp | BS087 | Used ofr dissolution |
Forceps | RWD | F12028 | Surgical instrument |
Hemostats | Biosharp | BS-HF-S-125 | Surgical instrument |
Isoflurane | RWD | 20071302 | Used for anesthesia |
Letrozole powder | Sigma | L6545-50MG | Primary acting drugs |
Needle and the 4-0 absorbable suture | JINGHUAN | CR413 | Surgical instrument |
Needle holder | ShangHaiJZ | J32010 | Surgical instrument |
Nitrile Gloves | Biosharp | BC040-L | Used for aseptic operation |
Osmotic Pumps | ALZET | 1004 | Letrozole storage and sustained release |
PEG(Poly(ethylene glycol)) | Solarbio | P8250 | Used ofr dissolution |
Physiological Saline Solution | Biosharp | BL158A | Mini-pump storage |
Pipette | Eppendorf | 3123000268-A | 100 μL-1000 μL |
Pipette | TopPette | 7010101008 | 10 μL-100 μL |
Povidone-iodine swabs | SingleLady | GB26368-2010 | Skin disinfection |
Scissors | Biosharp | BS-SOR-S-100P | Surgical instrument |
Small Animal Anesthesia Machine | RWD | R500IP | Used for anesthesia |
Sterile gauze | ZHENDE | BA69087 | Used for wiping liquids |
Syringe | Bofeng Biotech | BD300841 | 1 mL |
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