Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

Summary

We describe an in vitro assay to model chorioallantoic attachment, the first step in placenta formation. The protocol demonstrates the dissection and explant culture of murine allantoides on immobilized α4β1 integrin. Allantois attachment is evaluated microscopically at pre-determined time points.

Abstract

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental insults that disturb placenta development or function can cause early pregnancy loss in mice and humans. Nevertheless, simple in vitro assays to screen for potential effects on placenta formation are lacking. Here, we focus on modeling the first and critical step in placenta formation, which consists of the attachment of the allantois to the chorion. We describe a method to rapidly assess the attachment of allantoic explants on immobilized α4β1 integrin, which serves as a chorio-mimetic substrate.This in vitro approach enables a qualitative evaluation of the attachment and spreading behavior of multiple allantois explants at different consecutive time points. The protocol may be used to investigate the effect of targeted mouse mutations, drugs, or various environmental factors that have been linked to pregnancy complications or fetal loss on allantois attachment ex vivo.

Introduction

The placenta is indispensable for embryonic growth and development in the uterine environment. It constitutes the interface for oxygen, metabolite, and nutrient exchange between the fetal and maternal circulation, and also functions as an endocrine and immunological organ. Any endogenous or exogenous insult that impairs placental development can lead to intrauterine growth retardation, fetal loss, or pregnancy complications, both in mice and in humans¹.While the number of targeted mouse mutations that cause abnormal placental physiology continues to increase², with 219 genotypes currently listed on the Mouse Genome Infor....

Protocol

Mouse breeding was approved by the Regierung Unterfranken, and all analyses were carried out in strict accordance with all German and European Union applicable laws and regulations concerning care and use of laboratory animals.

1. Coating of Microtiter Plates with α4β1 Integrin for Ex Utero Allantois Explant Culture

1. Reconstitute the lyophilized murine α4β1 integrin at 200 µg/mL in sterile phosphate-buffered saline (PBS). Dilute to a final concentration of 10 µg/mL in pre-warmed (37 °C) PBS. Pipette 20 µL/well of the diluted α4β1 integrin solution into the required number....

Results

This protocol describes a method to isolate and explant murine allantoides ex vivo, and details the qualitative assessment of allantois attachment to α4β1 integrin, a critical process during in vivo chorioallantoic fusion. The representative results shown in Figure 1A-H demonstrate the successive steps of allantois isolation starting from the pregnant uterus. Maternal tissues that surround the uterine horns,.......

Discussion

In the presence of serum, which is a rich source of pro-adhesive extracellular matrix molecules such as fibronectin, allantois explants will readily attach to various plastic, glass, or filter surfaces^9,16. Thus, if the aim of the performed assay is to specifically investigate the effect of a genetic modification or any other treatment on allantois attachment to immobilized α4β1, it is critical to block non-specific binding sites (e.g., with bo.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
C57BL/6J wildtype mice	The Jackson Laboratory	Stock No: 000664
70 % (v/v) Ethanol	VWR International	930031006
Standard pattern forceps	Fine Science Tools	11000-12	Forceps used to open the abdominal cavity.
Micro dissecting forceps	Fine Science Tools	11254-20	Dumont # 5 medical biology forcepts with fine, sharp tip.
Fine scissors	Fine Science Tools	14094-11	Straight blades.
Spring scissors	Fine Science Tools	15012-12	Noyes spring scissors with 14 mm blades for the dissection of uterus buds.
Microspoon	Carl Roth	AT18.1	5 mm Spoon diameter.
Microtiter plates	ibidi	81501	We use uncoated, sterile angiogenesis slides (internal volume 50 µL) with a hydrophobic surface that is not tissue-culture treated to reduce non-α4β1-mediated binding to plastic.
10 cm dish	Nunc	150350	Sterile tissue culture dishes.
3.5 cm dish	Nunc	150288	Sterile tissue culture dishes.
Large orifice pipette tips	Biozym	VT0140X	Low binding pipette tips, 200 µL.
α4β1 integrin	R&D Systems	6054-A4	Murine α4β1 integrin recombinantly expressed and purified from a Chinese hamster ovary cell line.
Dulbecco's Phosphate Buffered Saline (PBS)	Sigma Aldrich	D8537	Without Ca2+ and Mg2+.
Dulbecco’s Modified Eagle Medium (DMEM)	PAN Biotech	P04-03600	The formulation contains 4.5 g/L glucose, 110 mg/L sodium pyruvate and 3.7 g/L NaHCO3 but no L-glutamine, which is added separately.
Penicillin/Streptomycin	Gibco (ThermoFisher Scientific)	15140-122	100 x (10,000 U/mL Penicillin and 10,000 µg/mL streptomycin) stock solution. Prepare and store aliquots at -20 °C to avoid freeze/thaw.
L-Glutamine	PAN Biotech	P04-80100	100 x (200 mM) Stock solution. Prepare and store aliquots at -20 °C to avoid freeze/thaw.
Fetal bovine serum	Biochrom	S 0115	We use heat-inactivated FBS (heated to 56 °C for 30 min with mixing to inactivate complement).
Bovine serum albumin (BSA)	Sigma Aldrich	A7030	We use protease- and fatty acid-free albumin. Prepare a 0.1 % (w/v) solution in DMEM. Sterile filter the solution through a disposable cell culture filter with a 0.22 µm pore size and low protein-binding membrane, and store aliquots at -20 °C.
para-Formaldehyde	Carl Roth	0335.2	To make a formaldehyde solution in PBS, weigh para-formaldehyde powder in a ventilated hood, and add it to PBS preheated to ca. 60 °C in a beaker on a stir plate. Add 1 N NaOH dropwise until solution clears. Let solution cool to room temperature, and filter through 0.22 mm filter syringe. Adjust pH with diluted HCl to ca. 6.9, and adjust to final volume with PBS. We aliquot and freeze the solution, but it can also be stored at 4 °C for approximately four weeks.
Triton X-100	Sigma Aldrich	X100	Use wide-orifice pipette tips to handle undiluted Triton X-100.
Normal goat serum	Sigma Aldrich	G9023	To block non-specific binding of antibodies in immunofluorescence analyses.
α-CD31 Antibodies	BD Biosciences	550274	Purified rat anti-mouse monoclonal antibodies, clone MEC 13.3.
Secondary goat anti-rat antibodies	Thermo Fisher	A-11006	Goat anti-rat IgG (heavy and light chains), cross-adsorbed, fluorescently labeled with Alexa Fluor 488. Other fluorescent labels are also possible.
Mowiol 4-88	Sigma Aldrich	81381	Mounting medium for cytochemistry. MW ~31,000
Labovert	Leitz	Labovert	Inverted phase contrast microscope equipped with a 4 x and 10 x objective for somite pair counting. Other phase contrast microscopes (such as those that are routinely used in tissue culture) are also suitable.
M80	Leica Microsystems	M80	Stereomicroscope with 8 : 1 zoom range (yielding a magnification between 7.5 x and 60 x) for embryo and allantois dissection.
DM4000B	Leica Microsystems	DM4000B	Microscope system for histology with LED illumination. We use HCX PL FLUOTAR 5 x/0.15 and HC PL FLUOTAR 10 x/0.30 objectives. Other microscopes equipped phase contrast and fluorescence optics can be used to score allantois attachment.
Digital camera	JVC	KY-F75U	3-CCD digital capture camera attached to the DM4000B LED microscope. We use Diskus software (Hilgers) to operate both the microscope and the camera.
Confocal microscope	Leica Microsystems	SP5	Confocal microscope for higher-resolution imaging of endothelia in the vascular plexus of allantois explants. Immunofluorescence images were taken with a 40 x objective.