Microinjection Method for Analyzing Zebrafish Early Stage Oocytes.

Manami Kobayashi,Mary C Mullins,Allison Jamieson-Lucy

doi:10.3389/fcell.2021.753642

Manami Kobayashi, Mary C Mullins + Show 1 more

Open Access

https://doi.org/10.3389/fcell.2021.753642

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Abstract

Maternal factors which accumulate and establish oocyte polarity during the early stages of oogenesis play key roles in embryonic development, as well as germ cell formation. However, vertebrate oogenesis, especially early stages of oogenesis, is not well understood due to the difficulty of accessing these oocytes and the lack of analytical methods. Here, we report on a microinjection method for analyzing zebrafish early-stage oocytes and some artifacts to be aware of when performing oocyte injections or analyzing oocytes. Using this method, we successfully injected mRNAs encoding fluorescent-tagged proteins into early-stage oocytes and observed subcellular localization in the live oocytes. This method is expected to advance the functional analysis of genes involved in oogenesis.

Highlights

Germ cells are specialized cells that differentiate into oocytes or sperm to propagate the generation
Vertebrate oogenesis has been little studied in part due to the inaccessibility of early stage oocytes in the ovary, as well as the lack of tools to manipulate these oocytes combined with more difficult genetic analysis
Mitotracker was used for staining mitochondria and the mitochondrial-rich structure called the Balbiani body (Bb) in stage 1 oocytes (Kosaka et al, 2007)

Summary

INTRODUCTION

Germ cells are specialized cells that differentiate into oocytes or sperm to propagate the generation. The first asymmetry found in stage I oocytes is the mitochondrial-rich structure called the Balbiani body, which is postulated to establish oocyte polarity (Dosch et al, 2004; Marlow and Mullins, 2008). This structure is conserved from insects to mammals (Jamieson-Lucy and Mullins, 2019). We provide a detailed protocol for microinjecting zebrafish early stage oocytes (Figure 1A) Using this method, we successfully injected early stage oocytes with mRNAs encoding fluorescent-tagged proteins and observed subcellular localization in the live oocytes (Figure 2). We recommend adding 100× Glutamax to 1× to L-15 medium without L-glutamine each time

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ETHICS STATEMENT