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

Summary

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.

Abstract

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.

Introduction

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 medicine^1,2.

Endometrial factors account for about two-thirds of the causes of IVF failure³. Human embryo implantation is divided into three stages: positioning, adhesion, and invasion^4,5,6. The maternal endometrium undergoes a series of changes to meet the arrival of the embryo. Forming an implantation "window period" provides favorable conditions for embryo implantation^7,8.

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 implantation^5,9,10. 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 functions^12,13. 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 elucidated^1,14.

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 decidualization^15,16. 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.

Protocol

All of the animal experiments described were approved by the Affiliated Drum Tower Hospital of Nanjing University Medical School'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 °C ± 1 °C, relative humidity of 50% ± 1%, a light/dark cycle of 12 h/12 h, and free access to food and water.

1. Mouse ovariectomy

1. Sterilize the surgical instruments with a high-temperature and high-pressure sterilizer 1 day before the operation.
2. Weigh 8-week-old C57BL/6 mice and intraperitoneally (i.p) inject 1% sodium pentobarbital accordingly to anesthetize the mice. The dose used is 40 mg/kg¹⁷. This dose provides 40-60 minutes of sedation, which meets the requirements of an ovariectomy. For pre-operative analgesia, inject mice with 5 mg/kg meloxicam (s.c).
   NOTE: Successful anesthesia in mice can be indicated by the disappearance of deep muscle reflexes and steady breathing. Signs of successful anesthesia include no blinking when touching the inner canthus, no swallowing when pulling the tongue, no leg bending when pinching the skin between the toes, and no bouncing when needling the tail.
3. Apply the protective eye ointment to the eyeballs to prevent dryness during the anesthesia.
4. Start the operation when mice are completely anesthetized. Place and fix 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 after shaving.
5. Find the operation incision site at 0.5 cm (or 1.0 cm below the rib) on the upper edge of the thigh root of both hind limbs parallel to the spine (Figure 1A).
   NOTE: A correct incision location is essential for quickly localizing the ovary.
6. Take the incision site as the center and disinfect the incision area with iodophor 3x, then place a surgical drape around the incision area.
7. Make a longitudinal incision of about 0.5-1.0 cm using scissors. Cut the fascia and passively separate the muscles with tweezers.
8. 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 (Figure 1B).
   NOTE: The white fat pad is the most obvious indicator of the location of the ovary.
9. Clamp the fat mass with hemostatic clips, and pull the ovary wrapped by the fat pad out of the incision.
10. Clamp the junction of the ovary and fallopian tube with curved tweezers, and remove the ovary along the ovarian pedicle with scissors. Stop the bleeding using an electrocoagulation pen or a needle of a 1 mL syringe heated on the alcohol lamp.
    NOTE: Be sure to preserve the fallopian tube, which is the anatomical landmark for the subsequent injection of sesame oil into the uterine horns.
11. Rinse the surgical incision with normal saline to prevent tissue adhesion, use a 4-0 suture to sew the muscle and skin respectively, and disinfect the surgical incision with iodophor again.
12. Place the mice in the prone position in the cage and resuscitate them in a 25 °C incubator equipped with a light source and a ventilation system for about 2 h.
13. 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.

2. Postoperative rest and estrogen and progesterone formulation

1. Make sure the mice rest for 2 weeks after ovariectomy.
2. In an ultra-clean cabinet, add estrogen (2 ng/µL) and progesterone (0.2 ng/µL) into sesame oil in RNA enzyme-free centrifuge tubes.
3. Place the centrifuge tubes in a thermostatic water bath at 37 °C and shake the tubes continuously to accelerate dissolution.
4. After complete dissolution, divide the sesame oil solution into 100 µL aliqouts for convenient use. Store the prepared estrogen and progesterone solutions in a refrigerator at 4 °C.

3. Induced artificial decidualization model

1. Subcutaneously (s.c) inject 100 ng of estrogen in 50 µL of sesame oil into each mouse for 3 days, followed by 2 days of rest¹⁵.
   NOTE: Ensure that the estrogen and progesterone have been formulated and placed in different places. Any contamination of estrogen by progesterone may lead to failure.
2. Inject (s.c.) 1 mg of progesterone and 10 ng of estrogen in 50 µL of sesame oil into each mouse for another 3 days¹⁵.
3. Operate the mice to induce the artificial decidualization model¹⁵ 6 h after the third estrogen-progesterone combined injection.
4. Find the uterus horn at the lower end of the fallopian tubes and inject 20 µL of sesame oil slowly along with the uterine horn, push the tissue back, suture the incision, and allow the mouse to recover. The approach to operation is the same as the mouse ovariectomy¹⁵. 
   NOTE: Only proceed with the second surgery in the event of a successful ovariectomy (skin incision is well healed, the ovary is completely excised, and the residual end of the fallopian tube is not adhered to the surrounding tissues).
   NOTE: For inducing the AD model, 20 µL of sesame oil is appropriate; more than 20 µL of sesame oil will easily penetrate the other side.
5. Inject (s.c.) 50 µL of sesame oil containing 1 mg of progesterone and 10 ng of estrogen (H.) for another 4 days. See details in Figure 2^15,18.

4. Sample collection

1. Euthanize the mice (n = 6) by carbon dioxide asphyxiation and collect the uteri. Observe the shape of the bilateral uteri, take photos, and weigh the uterine horns (Figure 3A, B).
2. Fix 3-5 mm of the uterus tissue with 10% formalin, embed in paraffin, and section them. Stain the sections with hematoxylin and eosin to observe the morphological changes¹⁹ (Figure 4A).
3. Embed another 3-5 mm of the uterus tissue in optimal cutting temperature compound (OCT), freeze in liquid nitrogen, and store in a −80 °C refrigerator. Frozen section the tissue to 10 µm and detect alkaline phosphatase activity in the uterine tissue by the azo coupling method²⁰ (Figure 4B).
   NOTE: Satisfactory results can be obtained using frozen sections to detect ALP content, not paraffin sections.
4. Extract the total RNAs of the uterus with Trizol reagent and perform quantitative real-time PCR (qPCR) on a real-time PCR system (Table of Materials) with qPCR master mix (Table of Materials) to detect the relative mRNA expression of prolactin family 8 subfamily member 2 (Prl8a2), alkaline phosphatase liver/bone/kidney (Alpl), and insulin-like growth factor-binding protein 1 (Igfbp1)¹⁵ by normalization to 18S mRNA levels (Figure 4C-E). Follow the PCR conditions: Stage 1, 95 °C for 30 s; Stage 2, 95 °C for 10 s and 60 °C for 30 s; repeat the cycle 40x. A complete list of primer sequences is provided in Table 1.
5. Analyze qPCR data using the 2^-ΔΔCT method and a t-test method for statistical analysis in the appropriate software. In this study, p < 0.05 was considered statistically significant.

Results

The mouse decidualization model indexes include the uterus'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...

Discussion

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

Materials

Name	Company	Catalog Number	Comments
Estrogen	Sigma	E2758	Hormone supplement
Progesterone	Sigma	P0130	Hormone supplement
Sesame oil	Sigma	S3547	Hormone supplement
Sodium pentobarbital	Dainippon Sumitomo Pharma Co.,Ltd.		Anaesthesia
Meloxicam injection	Qilu Animal Health Products Co., Ltd		Analgesia
Alkaline phophatase stain kit(kaplow's/azo coupling method)	Solarbio	G1480	Alkaline phophatase stain
Eosin	Servicebio	G1005-2	HE stain
Hematoxylin	Servicebio	G1005-1	HE stain
ChamQ Universal SYBR qPCR Master Mix	Vazyme	Q711-02	qPCR
70% ethanol	Lircon	ZH1120090	Disinfect
Iodophor	Runzekang	RZK-DF	Disinfect
Erythromycin Eye Ointment	Guangzhou Baiyunshan		Mice eyeball protect
4-0 suture	Ethicon	W329	Incision suture
10% formalin	Yulu	L25010118	Tissue fix
Optimal cutting temperature compound	Sakura	4583	Ssection
Trizol reagent	Ambion	15596018	qPCR