Home › Cell Biology › Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development
Cell Biology JoVE (Open Access) Citable · DOI

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

DOI: 10.3791/52496-v
What you'll learn
  • ✓ Manipulate and inject Xenopus laevis oocytes with oligonucleotides to modulate gene expression
  • ✓ Induce in vitro maturation of immature oocytes to mature eggs using hormone treatment
  • ✓ Perform intracytoplasmic sperm injection to generate embryos for developmental studies
  • ✓ Assess embryonic survival and developmental outcomes following ICSI in Xenopus
Protocol

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

Difficulty
advanced
Total time
~3–5 days (includes oocyte preparation, maturation induction, hormone timing, sperm collection, and ICSI)
Model organism
Xenopus laevis
Biosafety
BSL-1

Steps

1
Prepare oocytes and reagents for injection

Set up equipment, obtain Xenopus laevis oocytes, and prepare injection solutions and media. Establish sterile work conditions and calibrate micromanipulators.

▶ 01:14
2
Inject oocytes with oligonucleotides and induce maturation

Microinject oocytes with morpholino oligonucleotides or mRNA to alter gene expression, then trigger in vitro maturation using hormone stimulation. Monitor progression to metaphase II.

▶ 03:47
3
Perform intracytoplasmic sperm injection on mature eggs

Prepare sperm and inject a single sperm nucleus into the cytoplasm of each mature egg using a micromanipulator. Assess proper sperm integration and pronucleus formation.

▶ 05:55
4
Assess embryonic survival and developmental progression

Culture injected embryos and evaluate survival rates, morphology, and developmental stages. Compare outcomes between control and experimental conditions.

▶ 08:12
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