Name: A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome (Video) | JoVE Journal
Uploaded: 2018-10-02T14:30:00.000+00:00
Duration: 8 min 20 s
Description: Learn to create a lean PCOS-like mouse model using dihydrotestosterone pellets for reproductive biology research. This method enables consistent hormone release to study hyperandrogenism and its effects on offspring development.

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5 minutes

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.).

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 &#176;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
<ol>
	<li>DHT pellet preparation
	<ol>
		<li>Autoclave silastic tubing to sterilize. Cut silastic tubes to a length of 15 mm with a razor blade;</li>
		<li>Seal one side by injecting medical adhesive silicone into the tube with a 20 G&#160;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 Plunger should be withdrawn from the syringe and then adhesive inserted into the reservoir.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;</li>
		<li>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.</li>
		<li>Wear gloves, mask, goggles and lab coat before making DHT-pellet in a hood to avoid DHT exposure to skin.</li>
		<li>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.</li>
		<li>DHT powder can be tamped down with a large paper clip that is straightened.&#160; 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.</li>
		<li>Cut each sealed side to make the length of silicon 2 mm long. The total length of pellet will be 8 mm.</li>
		<li>Seal both side of empty silastic tubing as control (no-DHT) pellet.</li>
		<li>Keep pellets in a 50 mL conical tube wrapped with foil (to prevent light exposure) at room temperature until &#160;use. The DHT pellets maintain full efficacy for at least 3 months of storage.</li>
	</ol>
	</li>
	<li>DHT insertion and replacement
	<ol>
		<li>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 &#176;C for equilibration just before insertion.</li>
		<li>Prior to surgery, the surfaces and gloves are disinfected with clidox and the work surfaces are covered &#160;&#160;with clean plastic backed absorbent paper (surgical pad).&#160; All instruments that come in contact with animals are decontaminated prior to entering the animal facility by autoclaving.&#160; Instruments will be allowed to dry and cool prior to use.</li>
		<li>2-month-old female mice are used (4&#8211;5 mice/cage). Mice&#160;are injected intraperitoneally with Xylazine (3.5 mg/kg bw)&#160;and Ketamine (78.8 mg/kg bw) using an insulin syringe. Calculations for mixing and dosing anesthesia is in Table 1.&#160;</li>
		<li>After adequate anesthesia is achieved, as measured by loss of toe reflex and slowed breathing, the mouse will be prepped for surgery.</li>
		<li>Disinfect the skin of the area with betadine using sterile gauze and clean with 70% ethanol. &#160;The area will again be painted with betadine.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Test estrous cyclicity after 3 days of DHT insertion. Estrous cycle stage is assessed by vaginal cytology.&#160; Vaginal cells are collected for 16 consecutive days by using a p10 pipettor to squirt sterile saline (around 10 &#181;L) into the vaginal cavity and then withdrawing the saline with the same sterile pipette tip.&#160; Vaginal cells sloughed from vaginal wall mix with saline and are collected.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Put slides into staining solutions for 1 min each for buffer B&#160;and C. Wash slides with tap water and dry at room temperature.</li>
		<li>Examine cell morphology under a 10X objective light microscope.&#160; Cell morphology to distinguish proestrus (P), estrus (E), metestrus (M) and diestrus (D) is described in references27,28,29.</li>
		<li>Count the days in each stage and divided by the total number of days to calculate percent time at each stage.</li>
		<li>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&#160;&#181;L&#160;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.</li>
	</ol>
	</li>
	<li>Blood collection
	<ol>
		<li>Collect blood at various days after DHT insertion (we limit collection volume to 100 &#181;L for steroid and 30ul for LH/FSH assay) between 9 and 10 am by submandibular vein bleeding with lancet. This is described in11.</li>
		<li>Centrifuge blood at 6,000 x&#160;g at 4 &#730;C for 10 min, and collect the serum layer into another 1.5 mL tube that can be stored at -80 &#176;C until assay.</li>
	</ol>
	</li>
</ol>
2. Assess Reproductive Profiles of Female Offspring from Chronically DHT Inserted Dams
<ol>
	<li>Mating
	<ol>
		<li>The female mouse to be tested is moved into the cage with a proven fertile male (that has&#160; previously had pups with a female mouse) at 15 days following pellet insertion.&#160;</li>
		<li>Document dam&#8217;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.</li>
		<li>Collect blood of dams the second week after we observe increased body weight of dams greater than 3 g from the week before.&#160;</li>
	</ol>
	</li>
	<li>Puberty assessed by vaginal opening and first estrus
	<ol>
		<li>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.</li>
		<li>Once vagina opens, collect vaginal cells daily, as described in 1.2.8&#8211;1.2.11&#160;to check estrous cyclicity.</li>
		<li>Observe cell morphology as described 1.2.12, and the first estrus is defined as the date that all cells are cornified epithelial cells.&#160;</li>
	</ol>
	</li>
	<li>Body weight and ear punch
	<ol>
		<li>Dip the lancet tip into the tattoo paste and mark each pup with tattoo on the toe as&#160; &#160;described in reference12 at 7 PND, and weigh mice every 7 days until 70days of age.</li>
		<li>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.</li>
		<li>Collect blood at 21, 26, 70 PND after birth as described above in 1.3.1</li>
	</ol>
	</li>
	<li>Hormonal Assay
	<ol>
		<li>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.</li>
	</ol>
	</li>
</ol>

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<li>McNamara, K. M., Harwood, D. T., Simanainen, U., Walters, K. A., Jimenez, M., Handelsman, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measurement+of+sex+steroids+in+murine+blood+and+reproductive+tissues+by+liquid+chromatography-tandem+mass+spectrometry%5BTitle%5D)+AND+%22J.+Steroid+Biochem.+Mol.+Biol%22%5BJournal%5D)">Measurement of sex steroids in murine blood and reproductive tissues by liquid chromatography-tandem mass spectrometry.</a> J. Steroid Biochem. Mol. Biol. 121 (3-5), 611-618 (2010).</li>
<li>Klein, S. L., Bird, B. H., Glass, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sex+differences+in+Seoul+virus+infection+are+not+related+to+adult+sex+steroid+concentrations+in+Norway+rats%5BTitle%5D)+AND+%22J.+Virol%22%5BJournal%5D)">Sex differences in Seoul virus infection are not related to adult sex steroid concentrations in Norway rats.</a> J. Virol. 74 (17), 8213-8217 (2000).</li>
<li>Siracusa, M. C., Overstreet, M. G., Housseau, F., Scott, A. L., Klein, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((17beta-estradiol+alters+the+activity+of+conventional+and+IFN-producing+killer+dendritic+cells%5BTitle%5D)+AND+%22J.+Immunol%22%5BJournal%5D)">17beta-estradiol alters the activity of conventional and IFN-producing killer dendritic cells.</a> J. Immunol. 180 (3), 1423-1431 (2008).</li>
</ol>

Nothing to disclose.

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-...

Hyperandrogenism is the hallmark of polycystic ovary syndrome (PCOS) according to NIH criteria and of&#160;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&#160;exhibit alterations of basal serum estradiol, testosterone,&#160;LH and do not&#160;develop obesity, and&#160;show similar ovarian weight, serum levels of cholesterol, free fatty acids, leptin, TNF&#945; 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.

<table><tbody><tr><td>Crystalline 5&amp;alpha;-DHT powder</td><td>Sigma-Aldrich</td><td>A8380-1G</td><td></td></tr><tr><td>Dow Corning Silastic tubing</td><td>Fisher Scientific</td><td>11-189-15D</td><td>0.04in/1mm inner diameter x0.085 in/2.15 mm outer diameter</td></tr><tr><td>Medical adhesive silicone</td><td>Factor II, InC.</td><td>A-100</td><td></td></tr><tr><td>Goggles, lab coats, gloves and masks.</td><td></td><td></td><td></td></tr><tr><td>10 &amp;micro;L pipette tips without filter</td><td>USA Scientific</td><td>11113700</td><td></td></tr><tr><td>Microscope slide for smear</td><td>Fisher Scientific</td><td>12-550-003</td><td></td></tr><tr><td>Diff Quik for staining cells</td><td>Fisher Scientific</td><td>NC9979740</td><td></td></tr><tr><td>Lancet</td><td>Fisher Scientific</td><td>NC9416572</td><td></td></tr><tr><td>3 mL Syring</td><td>Becton, Dickinson and Company (BD),</td><td>30985</td><td></td></tr><tr><td>attached needle: 20 G</td><td>BD</td><td>305176</td><td></td></tr><tr><td>Ruler: any length than 10 cm with milimeter scale.</td><td></td><td></td><td></td></tr><tr><td>Xylazine</td><td>Vet one AnnSeA LA, MWI, Boise</td><td>NDC13985-704-10</td><td>100 mg/mL</td></tr><tr><td>Ketamine Hydrochloride</td><td>Hospira, Inc</td><td>NDC 0409-2051-05</td><td>100 mg/mL</td></tr><tr><td>Surgical staple</td><td>AutoClip&amp;reg; System, Fine Science Tool</td><td>12020-00</td><td></td></tr><tr><td>Insulin syringe</td><td>BD</td><td>329461</td><td>1/2 CC, low dose U-100 insulin syringe</td></tr><tr><td>Trocar</td><td>Innovative Research of America</td><td>MP-182</td><td></td></tr><tr><td>Microscope</td><td>Carl Zeiss Primo Star</td><td>415500-0010-001</td><td>Germany</td></tr><tr><td>Ear punch</td><td>Fisher Scientific</td><td>13-812-201</td><td></td></tr><tr><td>Testosterone rat/mouse ELISA kit</td><td>IBL</td><td>B79174</td><td></td></tr><tr><td>DHT ELISA kit</td><td>Alpha Diagnostic International</td><td>1940</td><td></td></tr><tr><td>One touch ultra glucometer</td><td>Life Scan, Inc.</td><td></td><td></td></tr><tr><td>One touch ultra test stripes</td><td>Life Scan, Inc.</td><td></td><td></td></tr><tr><td>Eppendorf tube</td><td>Fisher Scientific</td><td>05-402-18</td><td></td></tr><tr><td>Razor blade</td><td>Fisher Scientific</td><td>12-640</td><td></td></tr><tr><td>Clidox</td><td>Fisher Scientific</td><td>NC0089321</td><td></td></tr><tr><td>surgical underpad</td><td>Fisher Scientific</td><td>50587953</td><td>Manufacturer: Andwin Scientific 56616018</td></tr><tr><td>Betadine Antiseptic Solution</td><td>Walgreens</td><td></td><td></td></tr><tr><td>3M Vetbond (n-butyl cyanoacrylate)</td><td>3M Science. Applied to Life</td><td></td><td></td></tr></tbody></table>

a hyperandrogenic mouse model to study polycystic ovary syndrome

A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome

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.

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,...

We describe the development of a lean PCOS-like mouse model with&#160;dihydrotestosterone pellet to study the pathophysiology of PCOS and the offspring from these PCOS-like dams.

Hyperandrogenemia plays a critical role in reproductive and metabolic function in females and is the hallmark of polycystic ovary syndrome. Developing a ...

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Research

JoVE Journal

Developmental Biology

This article describes a way to prepare and insert dihydrotestosterone (DHT) pellets to model Polycystic Ovary Syndrome (PCOS) in mice. The example protocol demonstrates the procedure in mice used for reproductive and metabolic studies.

Subcutaneous Dihydrotestosterone (DHT) Treatment: Mouse Model of Polycystic Ovary Syndrome (PCOS)

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Whole-mount Confocal Microscopy for Adult Ear Skin: A Model System to Study Neuro-vascular Branching Morphogenesis and Immune Cell Distribution

Learn to create a lean PCOS-like mouse model using dihydrotestosterone pellets for reproductive biology research. This method enables consistent hormone release to study hyperandrogenism and its effects on offspring development.

A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome

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