Defining the Program of Maternal mRNA Translation during In vitro Maturation using a Single Oocyte Reporter Assay

Summary

This protocol describes a reporter assay to study the regulation of mRNA translation in single oocytes during in vitro maturation.

Abstract

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that take place in the cytoplasm in preparation for fertilization and acquisition of totipotency. During oocyte maturation, changes in gene expression depend exclusively on the translation and degradation of maternal messenger RNAs (mRNAs) rather than on transcription. Execution of the translational program, therefore, plays a key role in establishing oocyte developmental competence to sustain embryo development. This paper is part of a focus on defining the program of maternal mRNA translation that takes place during meiotic maturation and at the oocyte-to-zygote transition. In this method paper, a strategy is presented to study the regulation of translation of target mRNAs during in vitro oocyte maturation. Here, a Ypet reporter is fused to the 3' untranslated region (UTR) of the gene of interest and then micro-injected into oocytes together with polyadenylated mRNA encoding for mCherry to control for injected volume. By using time-lapse microscopy to measure reporter accumulation, translation rates are calculated at different transitions during oocyte meiotic maturation. Here, the protocols for oocyte isolation and injection, time-lapse recording, and data analysis have been described, using the Ypet/interleukin-7 (IL-7)-3' UTR reporter as an example.

Introduction

A fully-grown mammalian oocyte undergoes rapid changes in preparation for fertilization and acquisition of totipotency. These changes are essential to sustain embryonic development after fertilization. Although the events associated with nuclear maturation are relatively well described, much less is known about the molecular processes and pathways in the oocyte cytoplasm. During the final stages of oocyte maturation, oocytes are transcriptionally silent, and gene expression is entirely dependent on mRNA translation and degradation^1,2. The synthesis of proteins critical for developmental competence, therefore, ....

Protocol

The experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee of the University of California at San Francisco (protocol AN182026).

1. Preparation of media

1. 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 the day of use.

2. Preparation of mRNA encoding for Ypet-3' UTR and mCherry

1. Obt....

Results

Denuded prophase I-arrested oocytes of 21-day-old C57/BL6 mice were injected with a reporter mix containing mRNA encoding the Ypet reporter fused to the 3' UTR of IL-7 and mRNA encoding mCherry. YFP and mCherry expression was recorded in 39 oocytes, of which 30 were matured, and 9 were arrested in prophase I as a negative control. Three maturing oocytes were excluded for analysis because they either had a delayed GVBD (N=2) or moved in the dish during the recording (N=1). Figure 3 shows mChe.......

Discussion

The presented method describes a strategy to study activation and repression of translation of target mRNA at different transitions during in vitro oocyte meiotic maturation. IL-7, a cytokine released by the oocyte that may be involved in oocyte-cumulus cell communication^8,13, was chosen for the purpose of describing this method. IL-7 is known to be increasingly translated during oocyte maturation⁸ and allows for good visualizatio.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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