Name: generation of a mouse artificial decidualization model with ovariectomy for endometrial decidualization research
Uploaded: 2022-07-27T15:00:00
Description: Watch this Scientific Journal Video about Generation of a Mouse Artificial Decidualization Model with Ovariectomy for Endometrial Decidualization Research at JoVE.com

The authors wish to acknowledge support from the National Nature Science Foundation of China (82001629, XQS), the Youth Program of Natural Science Foundation of Jiangsu Province (BK20200116, XQS), and Jiangsu Province Postdoctoral Research Funding (2021K277B, XQS).

All of the animal experiments described were approved by the Affiliated Drum Tower Hospital of Nanjing University Medical School&#39;s Committee on the Use and Care of Animals (No. 20171202). All operations follow appropriate animal care and use agency and national guidelines.
NOTE: Mice were raised in a specific pathogen-free (SPF) environment, with a temperature of 22 &#176;C &#177; 1 &#176;C, relative humidity of 50% &#177; 1%, a light/dark cycle of 12 h/12 h, and free access to food and wa...

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Decidualization in mice is a spontaneous process depending on the presence of embryos, which is different from humans. However, it has been found that artificial stimulation such as uterine injection of glass beads and uterine laceration can induce decidualization of the endometrium instead of embryos. In addition, researchers found that many factors could induce decidual or participate in decidualization, such as the injection of steroid hormones, prostaglandins, and growth inhibitory factors into the uterine cavity<sup...

With the development of human-assisted reproductive technology, the current clinical pregnancy rate of in vitro fertilization-embryo transfer (IVF-ET) has reached or even exceeded that of natural pregnancy. Despite this, many patients in assisted reproduction clinical practice still undergo multiple embryo transfers but fail to achieve pregnancy as desired. However, its specific molecular mechanism is still unclear, so clinical intervention is ineffective, which is one of the significant challenges facing reproductive medicine1,2.
Endometrial factors account for about two-thirds of the causes of IVF failure3. Human embryo implantation is divided into three stages: positioning, adhesion, and invasion4,5,6. The maternal endometrium undergoes a series of changes to meet the arrival of the embryo. Forming an implantation &#34;window period&#34; provides favorable conditions for embryo implantation7,8.
In most mammals, after the blastocyst adheres to the luminal epithelium of the uterus, the stromal cells surrounding the blastocyst rapidly begin to proliferate and differentiate, and the rapid remodeling of the mesenchyme changes its shape and function, leading to embryo implantation5,9,10. The rapid increase in site volume and weight allows the blastocyst to become embedded in the uterine stroma, a process known as decidualization11. The endometrial stroma differentiates and remodels in preparation for pregnancy, while the transition of stromal cells provides space and new signaling connections for decidual cells to perform their functions12,13. Stromal cells transform into decidual cells and secrete many iconic factors such as prolactin (PRL), insulin-like growth factor-binding protein 1 (Igfbp1), and so on. Studies have shown that abnormal decidualization is one of the key reasons for embryo implantation failure, but the cause of abnormal decidualization is still unclear and needs to be further elucidated1,14.
The mouse artificial decidualization model is essential for studying the physiological process and molecular mechanisms underlying decidualization. Artificial decidualization (AD) mainly refers to the process of endometrial decidualization established by artificial methods to simulate pregnancy or the menstrual cycle. In terms of morphology, there is little overall difference between pregnancy decidualization and artificial decidualization15,16. The uterine glands exist in the endometrium before decidua form and disappear after decidualization. Regarding the gene expression, only a slight difference is identified between natural pregnancy decidualization (NPD) and AD15. Consequently, the artificial decidualization model in mice can simulate pregnancy decidualization to explore the unknown pathogenesis and new treatment of human reproductive diseases.
NPD, AD, and in vitro decidualization (IVD) are three methods to achieve mouse decidualization. The NPD model depends on natural pregnancy and is closest to the maternal physiological state, including the effects from embryos. Comparing the differences between implantation and non-implantation sites is a more physiological and convenient approach for studying decidualization. The AD model was developed by using an intrauterine injection of sesame oil as a stimulant to induce decidualization in a pseudopregnant female mouse mated with vasectomized males to avoid the impact from embryos. Both NPD and AD models play essential roles in different research purposes, but they cannot avoid mating failure and within-group differences caused by the different activities of maternal hormone metabolism. IVD is a method depending on the treatment of combined estrogen and progesterone at the cellular level, which requires more stringent experimental conditions and operating ability. However, the in vitro model cannot fully simulate the decidual response under physiological conditions15. Therefore, we propose a simple and improved induction method modified from traditional AD to reduce the effect of endogenous hormones on decidualization. Based on ensuring the success of decidualization induction, it is closer to the physiological state and more suitable for experiments that need to exclude embryo factors.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Estrogen</td>
			<td >Sigma</td>
			<td >E2758</td>
			<td >Hormone supplement</td>
		</tr>
		<tr >
			<td >Progesterone</td>
			<td >Sigma</td>
			<td >P0130</td>
			<td>Hormone supplement</td>
		</tr>
		<tr >
			<td >Sesame oil&#160;</td>
			<td >Sigma</td>
			<td>S3547</td>
			<td>Hormone supplement</td>
		</tr>
		<tr >
			<td >Sodium pentobarbital&#160;</td>
			<td >Dainippon Sumitomo Pharma Co.,Ltd.</td>
			<td ></td>
			<td>Anaesthesia</td>
		</tr>
		<tr >
			<td >Meloxicam injection</td>
			<td >Qilu Animal Health Products Co., Ltd</td>
			<td ></td>
			<td>Analgesia</td>
		</tr>
		<tr >
			<td >Alkaline phophatase stain kit(kaplow&#39;s/azo coupling method)</td>
			<td >Solarbio</td>
			<td>G1480</td>
			<td>Alkaline phophatase stain</td>
		</tr>
		<tr >
			<td >Eosin</td>
			<td >Servicebio</td>
			<td>G1005-2</td>
			<td>HE stain</td>
		</tr>
		<tr >
			<td >Hematoxylin</td>
			<td >Servicebio</td>
			<td >G1005-1</td>
			<td>HE stain</td>
		</tr>
		<tr >
			<td >ChamQ Universal SYBR qPCR Master Mix</td>
			<td >Vazyme</td>
			<td>Q711-02</td>
			<td>qPCR</td>
		</tr>
		<tr >
			<td >70% ethanol</td>
			<td >Lircon</td>
			<td>ZH1120090</td>
			<td>Disinfect</td>
		</tr>
		<tr >
			<td >Iodophor</td>
			<td >Runzekang</td>
			<td >RZK-DF</td>
			<td>Disinfect</td>
		</tr>
		<tr >
			<td >Erythromycin Eye Ointment</td>
			<td >Guangzhou Baiyunshan</td>
			<td ></td>
			<td>Mice eyeball protect</td>
		</tr>
		<tr >
			<td >4-0 suture</td>
			<td >Ethicon</td>
			<td >W329</td>
			<td>Incision suture</td>
		</tr>
		<tr >
			<td >10% formalin</td>
			<td >Yulu</td>
			<td >L25010118</td>
			<td>Tissue fix</td>
		</tr>
		<tr >
			<td >Optimal cutting temperature compound</td>
			<td >Sakura</td>
			<td >4583</td>
			<td>Ssection</td>
		</tr>
		<tr >
			<td >Trizol reagent</td>
			<td >Ambion</td>
			<td >15596018</td>
			<td>qPCR</td>
		</tr>
	</tbody>
</table>

generation of a mouse artificial decidualization model with ovariectomy for endometrial decidualization research

Endometrial decidualization is a unique differentiation process of the endometrium, closely related to menstruation and pregnancy. Impairment of decidualization leads to various endometrial disorders, such as infertility, recurrent miscarriage, and preterm birth. The development and use of the endometrial decidualization model in reproductive studies have been a highlight for reproductive researchers for a long time. The mouse has been extensively used in studying reproduction and decidualization. There are three well-established mouse models regarding decidualization, namely natural pregnancy decidualization (NPD), artificial decidualization (AD), and in vitro decidualization (IVD). Among them, AD is considered a reliable model for mouse decidualization, which is easy to implement and close to NPD. This paper focuses on a modified method of the generation and application process of the mouse artificial decidualization model with ovariectomy to avoid ovarian effects, which can obtain highly reproducible results with small within group variances. This method provides a good and reliable animal model for the study of endometrial decidualization.

The mouse decidualization model indexes include the uterus&#39;s general morphology, the mass ratio of the decidualized and non-decidualized uterus, the histological morphology of the endometrium, and the expression level of decidualization marker molecules. The general morphology of the artificial decidualized uterus of mice induced by oil is closer to that of the uterus in pregnancy. The uterine body becomes thick, and the uterine cavity becomes smaller than the non-induced side. The volume and weight of the induced ut...

Watch this Scientific Journal Video about Generation of a Mouse Artificial Decidualization Model with Ovariectomy for Endometrial Decidualization Research at JoVE.com

Generation of a Mouse Artificial Decidualization Model with Ovariectomy for Endometrial Decidualization Research

Here, we describe the method of generating an artificial decidualization model using the ovariectomized mouse, a classic endometrial decidualization experiment in the research field of endometrial decidualization.

Endometrial decidualization is a unique differentiation process of the endometrium, closely related to menstruation and pregnancy. Impairment of ...

Impairment of decentralization leads to virus of endometrial disorders such as in fatality, a recurrent miscarriage. This method provides a good and reliable animal model for the study of endometrial decentralization. This artificial decentralization model with Ovariectomy can avoid the influence of angelina over hormones which can often highly reproducible results with minor within group differences.To begin sterilize the surgical instruments with a high temperature and high pressure sterilizer one day before the operation. Apply the protective eye ointment to the eyeballs to prevent dryness during the anesthesia. Start the operation when mice are completely anesthetized.Place the mice in the prone position on the operating surface and shave the hair on the back after wetting them with soapy water. Disinfect the skin with 70%ethanol. Find the operation incision site at 0.5 centimeters on the upper edge of the thigh root of both hind limbs parallel to the spine.Take the incision site as the center and disinfect the incision area with iodophor, three times. Make a longitudinal incision of about 0.5 to one centimeter using scissors. Cut the fascia and passively separate the muscles with tweezers.Find a piece of the white fat pad in the incision field close to the lower pole of the kidney through the thin muscle layer. Clamp the fat mass with hemostatic clips and pull the ovary wrapped by the fat pad out of the incision. Clamp the junction of the ovary and fallopian tubes with curved tweezers and remove the ovary along the ovarian pedicle with scissors.Stop bleeding using an electrocoagulation pen or a needle of a one milliliter syringe heated on the alcohol lamp. Rinse the surgical incision with normal saline to prevent tissue adhesion. Use a 4-0 suture to sow the muscle and skin respectively and disinfect the surgical incision with iodophor again.Place the mice in the prone position in the cage and resuscitate them in a 25 degree Celsius incubator. Equipped with a light source and ventilation system for about two hours. Pay attention to the mice after the operation.Put them back in the original feeding place alone until they recover completely and give them enough water and food. Subcutaneously inject 100 nanograms of estrogen in 50 microliters of sesame oil into each mouse for three days followed by two days of rest. Inject one milligram of progesterone and 10 nanograms of estrogen in 50 microliters of sesame oil into each mouse for another three days.Operate the mice to induce the artificial residualization model six hours after the third estrogen progesterone combined injection. Find the uterus horn at the lower end of the fallopian tubes and inject 20 microliters of sesame oil slowly along the uterine horn. Push the tissue back, suture the incision and allow the mouse to recover.Fix three to five milliliters of the uterus tissue with 10%formalin embedded in paraffin and section them. Stain the sections with hematoxylin and eosin to observe the morphological changes. Embed another three to five millimeters of the uterus tissue in optimal cutting temperature compound freeze in liquid nitrogen and store in a minus 80 degree Celsius freezer.Frozen section the tissue in 10 micrometers and detect alkaline phosphatase activity in the uterine tissue by the Azo coupling method. The general morphology of the artificial decentralized uterus of mice induced by oil is closer to that of the uterus in pregnancy. The uterine body becomes thick and the uterine cavity becomes smaller than the non induced side.The volume and weight of the induced uterine horn are significantly higher than that of the non induced side. Hematoxylin and eosin staining of the uterine tissue showed that the glands disappear and the end endometrial stromal cells on the induced torn differentiated into large round cytoplasmic and multinucleated decidual cells with unclear cell boundaries. The non induced side sections show the endometrial tissue morphology under a normal physiological state.The result of the Azo coupling method showed that the alkaline phosphatase content of the oil induced side was significantly higher than that of the non induced side. The QPCR results showed that the mRNA expression of PRL-8-A-2, ALPL and IGFBP-1 in the induced horn was significantly higher than that in the non induced one, suggesting that oil can induce artificial decentralization in mice. Homo supplementation is a lengthy process that requires special attention contamination between homos which is the key to success.On the basis of this model uterine horn injection of Adenovirus can be performed which can study the relevant mechanism of specific molecules regulating residualization.

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