The In Vitro Single Oocyte Reporter Assay: A Technique to Study Target mRNA Translation Regulation During In Vitro Oocyte Maturation

Abstract

Source: Costermans, N. G. J. et al., Defining the Program of Maternal mRNA Translation during In vitro Maturation using a Single Oocyte Reporter Assay. J. Vis. Exp. (2021).

In this video, we demonstrate that in the presence of a phosphodiester inhibitor, the oocyte cell cycle is arrested at prophase-I, during which transcribed mRNAs are stored in the absence of translation. After the release of the inhibitor, oocyte maturation resumes, and the cells enter metaphase. The signal of reporter mRNA fused with the target gene in metaphase-I indicates the activation of translational machinery and protein expression.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Preparation of media Add all components, as described in Table 1, to make the basic oocyte collection medium and oocyte maturation medium. For the basic collection medium, set the pH to 7.4. For both collection and maturation medium, add 3 mg/mL of bovine serum albumin (BSA) and 1 µM cilostamide on…

Representative Results

Figure 1: Example of a single oocyte time-lapse recording. Brightfield, YFP, and mCherry recordings of a single oocyte injected with mRNAs encoding Ypet/Interleukin-7 3' UTR and polyadenylated mCherry at prophase I, MI (6 h after cilostamide release), and MII (15 h after cilostamide release). Scale bar = 25 µm. Abbreviations: YFP = yellow fluorescent protein; GV = germinal vesicle; MI = …

Materials

Preparation of media
Bovine Serum Albumin Powder Bioxtra	Sigma-Aldrich	SIAL-A3311
Cilostamide	EMD Millipore	231085
MEM alpha	Gibco	12561-056
Minimum Essential Medium Eagle	Sigma-Aldrich	M2645
Penicillin-Streptomycin 100x Solution, Sterile Filtered	Genesee Scientific Corporation (GenClone)	25-512
Sodium Bicarbonate	JT-Baker	3506-1
Sodium Pyruvate	Gibco	11360-070
Ultrapure distilled water	Invitrogen	10977-015
Preparation of mRNA encoding YFP/3' UTR and mCherry
Agarose	Apex Biomedical	20-102QD
Carbenicillin disodium salt	Sigma-Aldrich	C1389-1G
Choo-Choo Cloning Kit	McLab	CCK-20
CutSmart Buffer (10x)	New England Biolabs	B7204
DNA loading dye (6x)	Thermo Scientific	R0611
dNTP Solution	New England Biolabs	N0447S
DpnI	New England Biolabs	R0176
GeneRuler 1 kb DNA ladder	Thermo Fisher	SM1333
LB Agar Plates with 100 µg/mL Carbenicillin, Teknova	Teknova	L1010
LB Medium (Capsules)	MP Biomedicals	3002-021
MEGAclear Transcription Clean-Up Kit	Life Technologies	AM1908
MfeI-HF restriction enzyme	New England Biolabs	R3589
mMESSAGE mMACHINE T7 Transcription Kit	Invitrogen	AM1344
Phusion High Fidelity DNA polymerase	New England Biolabs	M0530
Poly(A) Tailing kit	Invitrogen	AM1350
QIAprep Spin Miniprep Kit	Qiagen	27106
QIAquick Gel Extraction Kit	Qiagen	28704
S.O.C. medium	Thermo Fisher	15544034
TAE buffer	Apex Biomedical	20-193
Ultrapure Ethidium Bromide Solution	Life Technologies	15585011
Oocyte collection
Aspirator tube assembly for calibrated micro-pipettes	Sigma-Aldrich	A5177-5EA
Calibrated micro-pipettes	Drummond Scientific Company	2-000-025
PMSG- 5000	Mybiosource	MBS142665
PrecisionGlide Needle 26 G x 1/2	BD	305111
Syringe 1 ml	BD	309659
Oocyte micro-injection
35 mm Dish | No. 0 Coverslip | 20 mm Glass Diameter | Uncoated	MatTek	P35G-0-20-C	For time-lapse microscopy
Borosilicate glass with filament	Sutter Instrument	BF100-78-10
Oil for Embryo Culture	Irvine Scientific	9305
Petri Dish	Falcon	351006	For micro-injection
Tissue Culture Dish	Falcon	353001	For oocyte incubation
VacuTip Holding Capillary	Eppendorf	5195000036
Software
Biorender	BioRender		Preparation of Figure 1S
MetaMorph, version 7.8.13.0	Molecular Devices		For time-lapse microscopy, analysis of 3' UTR translation