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 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.
Protocol
Representative Results
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….
Disclosures
The authors have nothing to disclose.
Acknowledgements
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).
Materials
| 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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