Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

Summary

To study the mechanism of lipid utilization in yolk sac membranes during the late stages of avian embryonic development, we established a primary Japanese quail embryonic endodermal epithelial cell culture system.

Abstract

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient utilization by avian embryos during development. Fertilized Japanese quail eggs were incubated at 37 °C for 5 days and then yolk sac membranes (YSM) were collected to establish the EEC culture system. We isolated the embryonic endoderm layer from YSM, and sliced the membrane into 2 - 3 mm pieces and partially digested with collagenase before seeding in 24-well culture plates. The EECs proliferate out of the tissue and are ready for cell culture studies. We found that the EECs had typical characteristics of YSM in vivo, for example, accumulation of lipid droplets, expression of sterol O-acyltransferase and lipoprotein lipase. The partial digestion treatment significantly increased the successful rate of EEC culture. Utilizing the EECs, we demonstrated that the expression of SOAT1 was regulated by the cAMP dependent protein kinase A related pathway. This primary Japanese quail EEC culture system is a useful tool to study embryonic lipid transportation and to clarify the role of genes involved in mediating nutrient utilization in YSM during avian embryonic development.

Introduction

The major nutritional resource of the avian embryo is yolk, composed of 33% lipids, 17% protein, and 1% ash.¹ During embryonic development, the yolk sac membrane (YSM) grows from within the embryonic abdominal cavity and gradually covers the yolk surface. Beginning at embryonic day 2, gene expression associated with lipid metabolism and angiogenesis is gradually increased in YSM, and the YSM slowly develops villus-like projections.^8,9 These projections increase absorption of yolk nutrients to support embryonic development. The YSM is an extraembryonic tissue that contains three germ layers, endoderm, mesoderm and ectoderm.¹⁴ The yolk s....

Protocol

NOTE: This procedure is a modification of a chicken model culture protocol developed by Bauer et al. 2013 and Nakazawa et al., 2011. ^2,9

1. Prepare Healthy Embryonic Day 5 Embryos from Japanese Quail

1. Place 1 male and 3 female sexually mature Japanese quail together in the same cage. Supply feed and water ad libitum. Adjust the light in the animal room to 14 hr of light and 10 hr of darkness.
2. Collect fertilized eggs in plastic bags every day in the afternoon and keep them in a 16 °C refrigerator to retard the growth rate of embryos.
3. After collecting a sufficient numbe....

Results

In order to achieve the goal of establishing a consistent and useful cell model, we need to extend and stabilize the proliferation rate and performance of avian EECs. We compared direct incubation of the endoderm with no enzyme digestion with endoderm partially digested with proteolytic enzymes, such as collagenase or collagenase plus 0.6 U of dispase. Dispase is an amino-endopeptidase that hydrolyzes the N-terminal peptide bonds of non-polar amino acid residues. Whereas protease digestio.......

Discussion

Because the previous culture system has only limited success, a better culture system is needed. The Japanese quail YSM endoderm requires treatment with proteolytic enzymes such as collagenase to loosen the cell-cell junctions to achieve better growth performance in the ex-vivo explants. Our data show that cell numbers from partial digestion treatment were greater than from the undigested tissue culture after seeding for 2 days (Figure 1). Therefore, the partial proteolytic digestion is a critical step t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco's Modified Eagle Medium	Gibco by Life technologies	12800-017 10 X 1 L	For wash the EECs pellets
D-MEM/F-12	Gibco by Life technologies	12400-024 10 X 1 L	As the basal medium in culturing EECs
NBCS	Gibco by Life technologies	16010-159	As the supplyment serum in culturing EECs
Pen-Strep Ampho. Solution	BI (Biological Industries)	03-033-1B 100 ml	For attenuating the possible infection
Collagenase Type IV	Gibco by Life technologies	17104-019 1 g	Collagenase is a protease with specificity for the bond between a neutral amino acid (X) and glycine in the sequence Pro-XGly-Pro. As the protease for dissociation of cells from primary tissue.
24-well plate	FALCON®	REF-353047	For EECs to attach and extension
50 ml PP centrifuge tubes	Corning® CentriStarTM	430829	For transportion of membranes and enzyme digestion
50 ml Conical bottomed Tube with Cap	PRO TECH	CT-50-PL-TW	For transportion of membranes and enzyme digestion
Reciprocal shaking bath	DEAGLE	SB302	For better enzymatic digestion on membranes