Isolation and In Vitro Decidualization of Mouse Primary Endometrial Stromal Cells

Summary

We aim to provide a method for the isolation and culturing of mouse endometrial stromal cells by enzymatic digestion. The isolated stromal cells are of high purity and can be decidualized by hormones in vitro. This protocol represents a feasible and stable method for stromal cell culture and for the further study of decidualization.

Abstract

A successful pregnancy relies on the establishment of decidualization, which involves the morphological and functional reprogramming of the progesterone-primed endometrial stromal cells under the influence of estrogen. In this protocol, we present a method for acquiring highly purified stromal cell isolated from the mouse uterus on day 4 of early pregnancy. Cultured primary stromal cells are then subject to further applications, such as RNA interference, overexpression, pharmaceutical treatment, immunoprecipitation, chromatic immunoprecipitation, and so on. Additionally, we provide a technique for the in vitro decidualization of cultured stromal cells using estrogen and progesterone. The in vitro decidualization method allows for the physically significant study of decidualization-related molecules. Altogether, this protocol provides a reliable and efficient method to facilitate further studies to define the molecular mechanism of decidualization.

Introduction

Decidualization is a prerequisite for the establishment of pregnancy. In mice, decidualization occurs after the attachment of the embryo to the uterine luminal epithelium during the receptivity stage¹. However, the mechanism underlying decidualization is still not completely understood². Considering the complex compositions of many cell types and the cell-specific expression of different genes in the uterus, there is a high demand for the purification of different cell types to investigate the potential function of each gene in different compartments.

The wall of the uterus consists of three l....

Protocol

All animal procedures were approved by the Animal Care and Use Committee of South China Agricultural University.

1. Preparation

1. Animals and treatments
   1. House adult mice (CD-1 strain) in a specific, pathogen-free room with a temperature- and light-controlled environment (12 h light/12 h dark).
   2. Mate mature female mice with fertile males at 16:00 h.
      NOTE: The day the vaginal plug is formed is considered day 1 of pregnancy.
   3. Sacrifice the mice by cervical dislocation.......

Discussion

The uterus, the residence of the conceptus, is of great importance for a successful pregnancy. A tight interaction between the conceptus and the maternal uterine endometrium initiates implantation and then the proliferation and differentiation of the stromal cells around the implantation sites, which will turn into specialized decidual cells under the influence of ovarian hormones. As a result of differentiation, decidual cells acquire various new functions during this transformation, such as providing a nutritional supp.......

Materials

Name	Company	Catalog Number	Comments
Hanks’ Balanced Salts	Sigma	H4891	For washing the tissues or cells
Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham	Sigma	D2906	For mESC culture
Trypsin	Amresco	0 458	For epithelial sheets dissociation
Dispase	Roche Applied Science	0 4942078001	For epithelial sheets dissociation
Collagenase I	Invitrogen	17100-017	For stromal cells dissociation
Charcoal Stripped  Foetal Bovine Serum	Biological Industries	04-201-1A	For the culture of mESC
Progesterone	Sigma	850454	For in vitro decidualization
Estradiol-17	Sigma	E1024	For in vitro decidualization
Vimentin	Cell Signaling Technology	3932s	Marker of stromal cells
0.22 μm Sterile Filter	Millipore	SLGP033RB	For filtration medium
CO2 Incubator	Thermo	3111	For the culture of mESC