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. Crea...

<ol>
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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.

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		<tr height="26">
			<td height="26" style="height:26px;width:243px;">Crystalline 5&#945;-DHT powder&#160;</td>
			<td style="width:135px;">&#160;Sigma-Aldrich</td>
			<td style="width:119px;">A8380-1G</td>
			<td style="width:412px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:243px;">Dow Corning Silastic tubing</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">11-189-15D</td>
			<td>0.04in/1mm inner diameter x0.085in/2.15mm outer diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Medical adhesive silicone</td>
			<td style="width:135px;">&#160;Factor II, InC.</td>
			<td style="width:119px;">&#160;A-100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Goggles, lab coats, gloves and masks.</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">&#160;10 &#181;L pipette tips without filter</td>
			<td style="width:135px;">USA Scientific</td>
			<td style="width:119px;">11113700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Microscope slide for smear</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">12-550-003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Diff Quik for staining cells</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">NC9979740</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">&#160; Lancet</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">NC9416572</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">3 mL Syring&#160;</td>
			<td style="width:135px;">Becton, Dickinson and Company (BD),</td>
			<td style="width:119px;">30985</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">&#160;attached needle: 20G</td>
			<td style="width:135px;">BD</td>
			<td style="width:119px;">305176</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">&#160;Ruler: any length than 10cm with milimeter scale.&#160;</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Xylazine&#160;</td>
			<td style="width:135px;">Vet one AnnSeA LA, MWI, Boise</td>
			<td style="width:119px;">NDC13985-704-10</td>
			<td>100mg/ml</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Ketamine Hydrochloride</td>
			<td style="width:135px;">Hospira, Inc</td>
			<td style="width:119px;">NDC 0409-2051-05</td>
			<td>100mg/ml</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">&#160;Surgical staple</td>
			<td style="width:135px;">&#160;AutoClip&#174; System, Fine Science Tool</td>
			<td style="width:119px;">12020-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">&#160;Insulin syringe</td>
			<td style="width:135px;">BD</td>
			<td style="width:119px;">329461</td>
			<td>1/2 CC, low dose U-100 insulin syringe</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:243px;">&#160;Trochar&#160;</td>
			<td style="width:135px;">Innovative Research of America</td>
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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&#8211;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,...

Watch this Scientific Journal Video about A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome at JoVE.com

A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome

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 ...

This method can help answer key questions in the field of reproduction. And the endocrinology. And are applicable to the study of Polycystic Ovary Syndrome or PCOS and hyperandrogenism.The advantage of this technic is that it facilitates a consistent, uniform, long-term release of hormones in a costly effective way. JHUs treating the procedure will be Ping Xue, a technician from my laboratory. For dihydrotestosterone or DHT pellet preparation, use a razor blade to cut a 15 millimeter piece of silicone tubing.And use a three milliliter syringe equipped with a blunted 20 gauge needle to inject two to five millimeters medical adhesive silicone into one end of the tube. Allow the tubing to dry overnight. And check for air bubbles on the sealed end.Wearing the appropriate personal protective equipment pour DHT powder into a plastic wayboat in the flowhood. Impress the opened end of the adhesive capped tube into the powder. Using a large straightened paperclip, tamp the powder down into the tubing, until the DHT reaches the appropriate experimental depth.Then seal the opened side of the tube with silicone overnight. The next morning, cut each sealed side to obtain a two millimeter length of silicone on both sides of the DHT and no DHT control pellets. Then store the pellets in the 50 milliliter conical tube wrapped with foil at room temperature for up to three months.For PCOS mouse model generation equilibrate up to 20 DHT or control pellets in separate 50 milliliter conical tubes containing 30 milliliters of 0.9 percent saline for 24 hours at 37 degrees Celsius. The next day, confirm lack of response to toe pinch in an anesthetized two month old female mouse and use sterile gauze to administer sequential Povidone-iodine and 70 percent ethynol scrubs to the surgical area. Next, make a five millimeter incision through the skin near the neck.And use a ten gauge trocar to make a 15 millimeter tunnel in the rostral direction. Use the trocar to insert a pellet dorsally into the tunnel and seal the incision with the surgical adhesive. Manually approximate the wound edges with forceps and use gentle brushing strokes to apply three thin film layers of liquid adhesive to one centimeter out from each side of the opposed wound edges.Then place the mouse alone in a cage on a heat pad with monitoring until full recumbency. Replacing the pellet every four weeks to maintain a constant level of androgen exposure. Three days after pellet insertion, use a p10 pipette to squirt ten microliters of sterile saline into the vaginal cavity and use the pipette tip to immediately recover the saline.Spread the vaginal cell saline sample onto a labeled slide. And allow the saline to dry at room temperature. Fix the dried slides in a container of 100 percent ethynol for at least five minutes.Then stain the slides for one minute each in buffer B and buffer C from a dif-quick staining kit. At the end of the buffer C incubation wash the slides with tap water for three seconds and dry them at room temperature. To determine the estrous cyclicity view the slides under the ten times objective of a light microscope.Count the days in each stage and divide by the total number of days to calculate percent time at each stage. To test the glucose tolerance, 20 days DHT insertion, fast the mice overnight. Weigh the mice the next morning, and use a glucometer and test strips to measure basil glucose level of tail vein blood.Inject the mice with two grams per kilogram body weight of 20 percent dextrose. Then measure the glucose level of tail vein blood samples at 15, 30, 60, 90, 120 minutes post dextrose administration. To acquire female offspring from DHT inserted dames, move the female mouse to tested in the cage with a proven fertile male at day 15, following pellet insertion.Document the dames body weight every week to determine if the mouse is pregnant. On post-natal day seven, dip a lancet tip into tattoo paste and mark each pup with a tattoo on the toe. Weigh the marked mice every seven days until 70 days of age.Between post-natal date 12 to 16, ear punch the mice to number them according to the system as illustrated in the figure. To distinguish the sex, examine the ventral side of each tagged mouse. The females will have visible nipples.To collect blood samples for various downstream analyses use a lancet to collect the approximate experimental volume of blood between nine and ten am by submandibular vein bleeding. And centrifuge the samples to allow serum collection. Then store the serums samples in 1.5 milliliter micro-centrifuge tubes at minus 80 degrees Celsius until their analysis.To assess puberty in the DHT female offspring, every day after weaning at post-natal day 21, check the vaginal opening by visual inspection. And use calibers to measure the ano-genital distance. Although the absolute values of serum DHT are different between mass spectrometry and Elisa analysis, the relative folk change of DHT versus no-DHT insertion is similar from both assays and across experiments.DHT levels are significantly increased from pre-conception through pregnancy in both DHT and no DHT mice. However, DHT levels are two-fold higher in DHT mice compared to no DHT mice at both pre-conceptional and gestational time points. Adult DHT daughters exhibit differences in reproductive function.For example, disrupted eestrous cyclicity compared to adult no DHT daughters experiencing significantly longer times in metestrus diestrus and less time in pro-esterous and esterous. While attempting this procedure it's important to remember to incubate the pellets for 24 hours before they are used for uniform release of the hormone. Following this procedure, the reproductive and metabolic consequences of DHT exposure can also be examined.After it's development, this technic paved the way for researchers in the field of endrochronology and maternal fetal interactions to study PCOS and the impact of low level hyperandrogenism in female mice. Don't forget that working with DHT can be extremely hazardous and the precautions such as wearing gloves and a mask and goggles, and working in a bio-safety hood, should always be taken while performing this procedure.

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Research

JoVE Journal

Developmental Biology

10.9K Görüntüleme.  Johns Hopkins University School of Medicine. Bu yöntem, çoğaltma alanındaki anahtar soruları yanıtlamaya yardımcı olabilir. Ve endokrinoloji. Polikistik Over Sendromu veya PKOS ve hiperandrojenizm çalışmaları için de geçerlidir.Bu tekniğin avantajı, pahalı etkili bir şekilde hormonların tutarlı, düzgün, uzun vadeli salınımını kolaylaştırıyor olmasıdır. Prosedürü tedavi eden JHUs Ping Xue, benim laboratuvar bir teknisyen olacaktır. Dihidrotestosteron veya DHT pelet hazırlama için, silikon boru 1...