Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Diego Marin; Alexandra L. Nguyen; Richard T. Scott, Jr.; Karen Schindler

doi:10.3791/57442

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Method Article

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

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DOI:

10.3791/57442

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April 10th, 2018

Diego Marin^*¹^,² , Alexandra L. Nguyen^*³ , Richard T. Scott, Jr.¹^,² , Karen Schindler³

¹IVI-RMA New Jersey, ²Jefferson College of Biomedical Sciences, Thomas Jefferson University, ³Department of Genetics, Rutgers, The State University of New Jersey

^* These authors contributed equally

Summary

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

Abstract

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

Introduction

Infertility is a condition that affects 10-15% of the human population of reproductive age ¹, from which nearly one-half seek medical treatment ². Although the etiology of infertility is diverse and in many cases multifactorial, the most common genetic abnormality in humans is embryonic aneuploidy ³. Aneuploidy is defined as the deviation (either gain or loss) of the correct number of chromosomes in a cell. The phenomenon of aneuploidy in human embryos is common and increases with advanced maternal age ⁴^,⁵. Four randomized controlled trial....