JoVE Logo
Authors
Contact Us

Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

We describe the development of a lean PCOS-like mouse model with dihydrotestosterone pellet to study the pathophysiology of PCOS and the offspring from these PCOS-like dams.

Abstract

Hyperandrogenemia plays a critical role in reproductive and metabolic function in females and is the hallmark of polycystic ovary syndrome. Developing a lean PCOS-like mouse model that mimics women with PCOS is clinically meaningful. In this protocol, we describe such a model. By inserting a 4 mm length of DHT (dihydrotestosterone) crystal powder pellet (total length of pellet is 8 mm), and replacing it monthly, we are able to produce a PCOS-like mouse model with serum DHT levels 2 fold higher than mice not implanted with DHT (no-DHT). We observed reproductive and metabolic dysfunction without changing body weight and body composition. While exhibiting a high degree of infertility, a small subset of these PCOS-like female mice can get pregnant and their offspring show delayed puberty and increased testosterone as adults. This PCOS-like lean mouse model is a useful tool to study the pathophysiology of PCOS and the offspring from these PCOS-like dams.

Introduction

Hyperandrogenism is the hallmark of polycystic ovary syndrome (PCOS) according to NIH criteria and of the Androgen Excess and PCOS (AE-PCOS) Society. Women with PCOS have difficulty getting pregnant and have increased risk of pregnancy complications1. Even if they get pregnant, their female offspring have an adverse health outcomes2,3. Animal models have been developed using various strategies4,5,6,7,8,9,10,11,12 and exhibiting many features of PCOS (anovulation, and or impaired glucose and insulin tolerance) with increased body weight and obesity associated with enlarged adipocyte size and increased adipocyte weight. There are two major strategies to produce animal models that are used to study PCOS. One is treatment with high levels of androgens directly (exogenous androgen injection/insertion) or indirectly (such as blocking androgen conversion to estrogen with aromatase inhibitor) after birth13. Another is by fetal hyperexposure of androgens during gestation14,15 to study the offspring. For example, female offspring from rhesus monkey16,17, sheep18, and rodents exposed to male levels of androgen during the intrauterine period develop PCOS-like traits later in life. These models significantly enhanced our understanding of elevated androgen effects, and fetal programing, and uterine environmental effects. However, these models have their own limitations: 1) animals develop obesity and it is therefore difficult to separate the effects of hyperandrogenemia from obesity induced reproductive and metabolic dysfunction; 2) before pregnancy, women with PCOS already exhibit high levels of androgen, thus oocytes have been exposed to androgen excess before fertilization; 3) the pharmacological doses of testosterone (T) or dihydrotestosterone (DHT) used after birth or during gestation may not reflect the androgen environment of PCOS. Testosterone and DHT levels have been measured in ovarian follicular fluid and/or serum, and testosterone and DHT levels are 1.5 to 3.9 fold higher in women with PCOS5,19,20,21,22,23 compared to unaffected women. We created an adult mouse model23,24,25 that develops reproductive and metabolic dysfunction within two weeks of the initiation of chronic DHT exposure from insertion of a pellet with 4mm length of crystal DHT powder (total length of pellet is 8mm). This model produces serum DHT levels that are about 2-fold higher (referred to as 2xDHT) than that of control mice without DHT treatment. The 2xDHT mice do not exhibit alterations of basal serum estradiol, testosterone, LH and do not develop obesity, and show similar ovarian weight, serum levels of cholesterol, free fatty acids, leptin, TNFα and IL-623,24,25 relative to controls even up to 3.5 months after DHT insertion23,24,25. Additionally, by mating females that have already developed features of PCOS, we can study the impact of a hyperandrogenic maternal environment on the metabolic and reproductive health of the offspring15.

This new paradigm (relevant to NIH and AE-PCOS Society criteria) models the disease by producing relatively similar levels of androgens to those of women with PCOS 2- to 3-fold higher testosterone or DHT levels compared to unaffected women. However, this model is maintained by continual exogenous DHT and not from programmed endogenous hyperandrogenism once DHT is withdrawn. The overall goal of this article is to focus on 1) how to make the DHT pellet; 2) how to generate a lean-PCOS like mouse model; 3) strategies to evaluate female offspring from these dams. Other measurements and assessment of phenotypes are not addressed in this manuscript but can be found in5,15,23,24,25,26.

Access restricted. Please log in or start a trial to view this content.

Protocol

Here, we present detailed protocols for DHT pellet preparation and insertion, and for reproductive and metabolic testing. The mice used in this study were a mixed background (C57/B6, CD1, 129Sv) and were maintained with food and water ad libitum in a 14/10 h light/dark cycle at 24 °C in the Broadway Research Building animal facility at the Johns Hopkins University School of Medicine. All procedures were approved by the Johns Hopkins University Animal Care and Use Committee.

1. Create PCOS-like Mouse Model

  1. DHT pellet preparation
    1. Autoclave silastic tubing to sterilize. Cut silastic tubes to a length of 15 mm with a razor blade;
    2. Seal one side by injecting medical adhesive silicone into the tube with a 20 G blunted needle attached to a 3 mL syringe. Plunger should be withdrawn from the syringe and then adhesive inserted into the reservoir. The plunger is reinserted and adhesive pushed down until it begins to emerge from the needle. The blunted needle is made by cutting the sharp tip side of 20 G needle with any strong scissors. The length of silicon in the tube should be more than 2 mm to allow for post-production trimming;
    3. Dry overnight; check for air bubbles on the sealed side. Those with no air bubbles are used for DHT pellets. Others can be used for no-DHT control pellets.
    4. Wear gloves, mask, goggles and lab coat before making DHT-pellet in a hood to avoid DHT exposure to skin.
    5. Pour DHT powder into a plastic weigh boat and press the open side of the adhesive capped tubes (produced in A1.1) into the DHT powder.
    6. DHT powder can be tamped down with a large paper clip that is straightened.  Continue until DHT reaches a height of 4 mm (or desired length). Check the length of DHT with a ruler, and seal the open side with silicon, dry overnight.
    7. Cut each sealed side to make the length of silicon 2 mm long. The total length of pellet will be 8 mm.
    8. Seal both side of empty silastic tubing as control (no-DHT) pellet.
    9. Keep pellets in a 50 mL conical tube wrapped with foil (to prevent light exposure) at room temperature until  use. The DHT pellets maintain full efficacy for at least 3 months of storage.
  2. DHT insertion and replacement
    1. Up to 20 DHT pellets or control pellets are submerged separately in a 50 mL conical tube with 30 mL sterile 0.9% saline for 24 h at 37 °C for equilibration just before insertion.
    2. Prior to surgery, the surfaces and gloves are disinfected with clidox and the work surfaces are covered   with clean plastic backed absorbent paper (surgical pad).  All instruments that come in contact with animals are decontaminated prior to entering the animal facility by autoclaving.  Instruments will be allowed to dry and cool prior to use.
    3. 2-month-old female mice are used (4–5 mice/cage). Mice are injected intraperitoneally with Xylazine (3.5 mg/kg bw) and Ketamine (78.8 mg/kg bw) using an insulin syringe. Calculations for mixing and dosing anesthesia is in Table 1. 
    4. After adequate anesthesia is achieved, as measured by loss of toe reflex and slowed breathing, the mouse will be prepped for surgery.
    5. Disinfect the skin of the area with betadine using sterile gauze and clean with 70% ethanol.  The area will again be painted with betadine.
    6. Cut a hole around 5 mm length with scissors under the skin near the neck. Using a 10 G trochar, make a small tunnel (15 mm) in the rostral direction. The pellet is inserted dorsally with the trochar.
    7. The opening is then sealed with surgical adhesive. Manually approximate the wound edges with forceps and gentle brushing strokes to apply a thin film of liquid adhesive to the approximated wound edges. Buildup 3 thin layers of adhesive to ensure the adhesive is evenly distributed over the wound. The adhesive should extend 1 cm on each side of the apposed wound edges. Suture or surgical clips can also be used to close the hole. Put mice back into cage, individually housed, with a heat pad for recovery. Replace pellet every 4 weeks to maintain a constant level of androgen exposure. The original pellet will be removed and new pellet will be inserted as described in A1.15 in the similar position.
    8. Test estrous cyclicity after 3 days of DHT insertion. Estrous cycle stage is assessed by vaginal cytology.  Vaginal cells are collected for 16 consecutive days by using a p10 pipettor to squirt sterile saline (around 10 µL) into the vaginal cavity and then withdrawing the saline with the same sterile pipette tip.  Vaginal cells sloughed from vaginal wall mix with saline and are collected.
    9. Spread saline with cells onto a labeled slide. Each slide can contain six samples. Slides are labeled to note which sample is placed in each of the six locations.
    10. After the saline has completely dried out on the slide, put slides into a container with 100% ethanol to fix cells. Slides should be fixed for at least 5 min, but can remain in fixative indefinitely until further use.
    11. Put slides into staining solutions for 1 min each for buffer B and C. Wash slides with tap water and dry at room temperature.
    12. Examine cell morphology under a 10X objective light microscope.  Cell morphology to distinguish proestrus (P), estrus (E), metestrus (M) and diestrus (D) is described in references27,28,29.
    13. Count the days in each stage and divided by the total number of days to calculate percent time at each stage.
    14. Test glucose tolerance at 21 days after DHT insertion by fasting the mice overnight (16 h) and injecting 2 g/kg body weight of 20% dextrose intraperitoneally. For example, if a mouse body weight is 25 g, this mouse will be injected with 250 µL of 20% dextrose with a 50 cc (0.5 mL) insulin syringe. Glucose levels are assessed by glucometer and test strips by sampling blood from tail vein at 0, 15, 30, 60, 90, 120 min. This is described in detail elsewhere30.
  3. Blood collection
    1. Collect blood at various days after DHT insertion (we limit collection volume to 100 µL for steroid and 30ul for LH/FSH assay) between 9 and 10 am by submandibular vein bleeding with lancet. This is described in11.
    2. Centrifuge blood at 6,000 x g at 4 ˚C for 10 min, and collect the serum layer into another 1.5 mL tube that can be stored at -80 °C until assay.

2. Assess Reproductive Profiles of Female Offspring from Chronically DHT Inserted Dams

  1. Mating
    1. The female mouse to be tested is moved into the cage with a proven fertile male (that has  previously had pups with a female mouse) at 15 days following pellet insertion. 
    2. Document dam’s body weight every week to determine if the mouse is pregnant. Dam weight increases of more than 3 g in a week indicates pregnancy.
    3. Collect blood of dams the second week after we observe increased body weight of dams greater than 3 g from the week before. 
  2. Puberty assessed by vaginal opening and first estrus
    1. Check vaginal opening by visual inspection every day after pups are weaned at 21 post-natal day (PND), measure the anogenital distance and record the age of vaginal opening.
    2. Once vagina opens, collect vaginal cells daily, as described in 1.2.8–1.2.11 to check estrous cyclicity.
    3. Observe cell morphology as described 1.2.12, and the first estrus is defined as the date that all cells are cornified epithelial cells. 
  3. Body weight and ear punch
    1. Dip the lancet tip into the tattoo paste and mark each pup with tattoo on the toe as   described in reference12 at 7 PND, and weigh mice every 7 days until 70days of age.
    2. Ear punch mice to number them using system in Figure 1 between 12 to 16 PND. To distinguish sex, examine ventral side of body, females will have visible nipples.
    3. Collect blood at 21, 26, 70 PND after birth as described above in 1.3.1
  4. Hormonal Assay
    1. DHT levels in blood serum are measured by both enzyme-linked immunosorbent assay (ELISA) and by liquid chromatography tandem mass spectrometry (LC-MS)23,24,31,32. T is measured by LC-MS, or a rat/mouse ELISA that has been validated by the University of Virginia Ligand Assay Core in the Center for Research in Reproduction23,24.

Access restricted. Please log in or start a trial to view this content.

Results

Serum DHT levels and Glucose tolerance test

DHT levels were measured from collected serum by both ELISA and by LC-MS according to protocol 1.24–1.25, and 2.9, 3.0. The DHT absolute values are different between mass spectrometry and ELISA, however, the relative fold (around 2-fold) of DHT vs no-DHT insertion is similar from both assays and across experiments15,23,...

Access restricted. Please log in or start a trial to view this content.

Discussion

Hyperandrogenism is a key feature of PCOS. The serum DHT levels (two fold higher in DHT mice than in no-DHT mice) used in this protocol are lower than those reported by other investigators in previous studies and are calibrated to proportionally mimic women with PCOS5,19,20,21. Unlike other models, this 2-fold DHT model does not alter the body weight and whole body composition compared with no-...

Access restricted. Please log in or start a trial to view this content.

Disclosures

Nothing to disclose.

Acknowledgements

This work was supported by the National Institutes of Health (Grants R00-HD068130 to S.W.) and the Baltimore Diabetes Research Center: Pilots and Feasibility Grant (to S.W.).

Access restricted. Please log in or start a trial to view this content.

Materials

NameCompanyCatalog NumberComments
Crystalline 5α-DHT powderSigma-AldrichA8380-1G
Dow Corning Silastic tubingFisher Scientific11-189-15D0.04in/1mm inner diameter x0.085 in/2.15 mm outer diameter
Medical adhesive siliconeFactor II, InC.A-100
Goggles, lab coats, gloves and masks.
10 µL pipette tips without filterUSA Scientific11113700
Microscope slide for smearFisher Scientific12-550-003
Diff Quik for staining cellsFisher ScientificNC9979740
LancetFisher ScientificNC9416572
3 mL SyringBecton, Dickinson and Company (BD),30985
attached needle: 20 GBD305176
Ruler: any length than 10 cm with milimeter scale.
XylazineVet one AnnSeA LA, MWI, BoiseNDC13985-704-10100 mg/mL
Ketamine HydrochlorideHospira, IncNDC 0409-2051-05100 mg/mL
Surgical stapleAutoClip® System, Fine Science Tool12020-00
Insulin syringeBD3294611/2 CC, low dose U-100 insulin syringe
TrocarInnovative Research of AmericaMP-182
MicroscopeCarl Zeiss Primo Star415500-0010-001Germany
Ear punchFisher Scientific13-812-201
Testosterone rat/mouse ELISA kitIBLB79174
DHT ELISA kitAlpha Diagnostic International1940
One touch ultra glucometerLife Scan, Inc.
One touch ultra test stripesLife Scan, Inc.
Eppendorf tubeFisher Scientific05-402-18
Razor bladeFisher Scientific12-640
ClidoxFisher ScientificNC0089321
surgical underpadFisher Scientific50587953Manufacturer: Andwin Scientific 56616018
Betadine Antiseptic SolutionWalgreens
3M Vetbond (n-butyl cyanoacrylate)3M Science. Applied to Life

References

  1. Palomba, S., de Wilde, M. A., Falbo, A., Koster, M. P., La Sala, G. B., Fauser, B. C. Pregnancy complications in women with polycystic ovary syndrome. Hum. Reprod. Update. 21 (5), 575-592 (2015).
  2. Doherty, D. A., Newnham, J. P., Bower, C., Hart, R. Implications of polycystic ovary syndrome for pregnancy and for the health of offspring. Obstet. Gynecol. 125 (6), 1397-1406 (2015).
  3. de Wilde, M. A., et al. Cardiovascular and Metabolic Health of 74 Children From Women Previously Diagnosed With Polycystic Ovary Syndrome in Comparison With a Population-Based Reference Cohort. Reprod. Sci. , 1933719117749761 (2018).
  4. Caldwell, A. S., et al. Characterization of reproductive, metabolic, and endocrine features of polycystic ovary syndrome in female hyperandrogenic mouse models. Endocrinology. 155 (8), 3146-3159 (2014).
  5. van Houten, E. L., Kramer, P., McLuskey, A., Karels, B., Themmen, A. P., Visser, J. A. Reproductive and metabolic phenotype of a mouse model of PCOS. Endocrinology. 153 (6), 2861-2869 (2012).
  6. Cardoso, R. C., Puttabyatappa, M., Padmanabhan, V. Steroidogenic versus Metabolic Programming of Reproductive Neuroendocrine, Ovarian and Metabolic Dysfunctions. Neuroendocrinology. 102 (3), 226-237 (2015).
  7. Dumesic, D. A., Abbott, D. H., Padmanabhan, V. Polycystic ovary syndrome and its developmental origins. Rev. Endocr. Metab Disord. 8 (2), 127-141 (2007).
  8. Kauffman, A. S., et al. A Novel Letrozole Model Recapitulates Both the Reproductive and Metabolic Phenotypes of Polycystic Ovary Syndrome in Female Mice. Biol Reprod. 93 (3), 69(2015).
  9. Kelley, S. T., Skarra, D. V., Rivera, A. J., Thackray, V. G. The Gut Microbiome Is Altered in a Letrozole-Induced Mouse Model of Polycystic Ovary Syndrome. PLoS One. 11 (1), e0146509(2016).
  10. Kafali, H., Iriadam, M., Ozardali, I., Demir, N. Letrozole-induced polycystic ovaries in the rat: a new model for cystic ovarian disease. Arch. Med. Res. 35 (2), 103-108 (2004).
  11. Maliqueo, M., Benrick, A., Stener-Victorin, E. Rodent models of polycystic ovary syndrome: phenotypic presentation, pathophysiology, and the effects of different interventions. Semin. Reprod. Med. 32 (3), 183-193 (2014).
  12. Yanes, L. L., et al. Cardiovascular-renal and metabolic characterization of a rat model of polycystic ovary syndrome. Gend. Med. 8 (2), 103-115 (2011).
  13. Kauffman, A. S., et al. A Novel Letrozole Model Recapitulates Both the Reproductive and Metabolic Phenotypes of Polycystic Ovary Syndrome in Female Mice. Biol. Reprod. 93 (3), 69(2015).
  14. Filippou, P., Homburg, R. Is foetal hyperexposure to androgens a cause of PCOS? Hum. Reprod. Update. 23 (4), 421-432 (2017).
  15. Wang, Z., Shen, M., Xue, P., DiVall, S. A., Segars, J., Wu, S. Female Offspring From Chronic Hyperandrogenemic Dams Exhibit Delayed Puberty and Impaired Ovarian Reserve. Endocrinology. 159 (2), 1242-1252 (2018).
  16. Abbott, D. H., Barnett, D. K., Bruns, C. M., Dumesic, D. A. Androgen excess fetal programming of female reproduction: a developmental aetiology for polycystic ovary syndrome? Hum. Reprod. Update. 11 (4), 357-374 (2005).
  17. Abbott, D. H., Dumesic, D. A., Franks, S. Developmental origin of polycystic ovary syndrome - a hypothesis. J. Endocrinol. 174 (1), 1-5 (2002).
  18. Padmanabhan, V., Veiga-Lopez, A. Sheep models of polycystic ovary syndrome phenotype. Mol. Cell. Endocrinology. 373 (1-2), 8-20 (2013).
  19. Pierre, A., et al. Dysregulation of the Anti-Mullerian Hormone System by Steroids in Women With Polycystic Ovary Syndrome. J. Clin. Endocrinol. Metab. 102 (11), (2017).
  20. Dumesic, D. A., et al. Hyperandrogenism Accompanies Increased Intra-Abdominal Fat Storage in Normal Weight Polycystic Ovary Syndrome Women. J. Clin. Endocrinol. Metab. 101 (11), 4178-4188 (2016).
  21. Fassnacht, M., Schlenz, N., Schneider, S. B., Wudy, S. A., Allolio, B., Arlt, W. Beyond adrenal and ovarian androgen generation: Increased peripheral 5 alpha-reductase activity in women with polycystic ovary syndrome. J. Clin. Endocrinol. Metab. 88 (6), 2760-2766 (2003).
  22. Dikensoy, E., Balat, O., Pence, S., Akcali, C., Cicek, H. The risk of hepatotoxicity during long-term and low-dose flutamide treatment in hirsutism. Arch. Gynecol. Obstet. 279 (3), 321-327 (2009).
  23. Ma, Y., et al. Androgen Receptor in the Ovary Theca Cells Plays a Critical Role in Androgen-Induced Reproductive Dysfunction. Endocrinology. , en20161608(2016).
  24. Andrisse, S., et al. Low Dose Dihydrotestosterone Drives Metabolic Dysfunction via Cytosolic and Nuclear Hepatic Androgen Receptor Mechanisms. Endocrinology. , en20161553(2016).
  25. Andrisse, S., Billings, K., Xue, P., Wu, S. Insulin signaling displayed a differential tissue-specific response to low-dose dihydrotestosterone in female mice. Am. J. Physiol.Endocrinol. Metab. 314 (4), E353-E365 (2018).
  26. van Houten, E. L., Visser, J. A. Mouse models to study polycystic ovary syndrome: a possible link between metabolism and ovarian function? Reprod. Biol. 14 (1), 32-43 (2014).
  27. Caligioni, C. S. Assessing reproductive status/stages in mice. Curr. Protoc. Neurosci. , Appendix 4, Appendix 4I (2009).
  28. Wu, S., et al. Conditional knockout of the androgen receptor in gonadotropes reveals crucial roles for androgen in gonadotropin synthesis and surge in female mice. Mol. Endocrinol. 28 (10), 1670-1681 (2014).
  29. Nelson, J. F., Felicio, L. S., Randall, P. K., Sims, C., Finch, C. E. A longitudinal study of estrous cyclicity in aging C57BL/6J mice: I. Cycle frequency, length and vaginal cytology. Biol. Reprod. 27 (2), 327-339 (1982).
  30. Dinger, K., et al. Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes. Journal of Visualized Experiments. (133), (2018).
  31. Nilsson, M. E., et al. Measurement of a Comprehensive Sex Steroid Profile in Rodent Serum by High-Sensitive Gas Chromatography-Tandem Mass Spectrometry. Endocrinology. 156 (7), (2015).
  32. McNamara, K. M., Harwood, D. T., Simanainen, U., Walters, K. A., Jimenez, M., Handelsman, D. J. Measurement of sex steroids in murine blood and reproductive tissues by liquid chromatography-tandem mass spectrometry. J. Steroid Biochem. Mol. Biol. 121 (3-5), 611-618 (2010).
  33. Klein, S. L., Bird, B. H., Glass, G. E. Sex differences in Seoul virus infection are not related to adult sex steroid concentrations in Norway rats. J. Virol. 74 (17), 8213-8217 (2000).
  34. Siracusa, M. C., Overstreet, M. G., Housseau, F., Scott, A. L., Klein, S. L. 17beta-estradiol alters the activity of conventional and IFN-producing killer dendritic cells. J. Immunol. 180 (3), 1423-1431 (2008).

Access restricted. Please log in or start a trial to view this content.

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Explore More Articles

Polycystic Ovary SyndromePCOSHyperandrogenismDihydrotestosteroneDHTMouse ModelSilicone TubingPellet ImplantationSurgical ProcedureAndrogen Exposure

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Contact Us

Recommend to library

JoVE NEWSLETTERS

Research

Education

Authors

Librarians

ABOUT JoVE

Copyright © 2025 MyJoVE Corporation. All rights reserved