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Mouse Oocyte Microinjection, Maturation and Ploidy Assessment
Published on: July 23, 2011
Down-regulation of the inositol 1,4,5-trisphosphate receptor in mouse eggs following fertilization or parthenogenetic
1Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts, 01003, USA.
Abstract:
Fertilization in mammalian eggs is characterized by the presence of intracellular calcium ([Ca(2+)]i) oscillations. In mouse eggs, these oscillations cease after a variable period of time and this is accompanied by a decrease in inositol 1,4,5-trisphosphate receptor (IP3R) responsiveness and down-regulation of the IP3R type 1 (IP3R-1). To investigate the signaling pathway responsible for inducing IP3R-1 down-regulation during fertilization, mouse eggs were exposed to or injected with several Ca(2+)-releasing agonists and the amounts of IP3R-1 immunoreactivity evaluated by Western blotting. Exposure to ethanol or ionomycin, which induce a single [Ca(2+)]i rise, failed to signal down-regulation of IP3R-1. However, [Ca(2+)]i oscillations induced by injection of boar sperm fractions (SF), which presumably stimulate production of IP3, or adenophostin A, an IP3R agonist, both induced down-regulation of IP3R-1 of a magnitude similar to or greater than that observed after fertilization. Exposure to thimerosal, an oxidizing agent that modifies the IP3R without stimulating production of IP3, also initiated down-regulation of IP3R-1, although oscillations initiated by SrCl(2) failed to evoke down-regulation of IP3R-1. The degradation of IP3R-1 in mouse eggs appears to be mediated by the proteasome pathway because it was inhibited by preincubation with lactacystin, a very specific proteasome inhibitor. We therefore suggest that persistent stimulation of the phosphoinositide pathway in mouse eggs by the sperm during fertilization or by injection of SF leads to down-regulation of the IP3R-1.
Insights
Fertilization in mouse eggs involves calcium oscillations that lead to down-regulation of inositol 1,4,5-trisphosphate receptor type 1 (IP3R-1). Persistent phosphoinositide pathway stimulation triggers this IP3R-1 degradation via the proteasome pathway.
Area of Science:
- Reproductive Biology
- Cell Signaling
- Molecular Biology
Background:
- Mammalian egg fertilization is marked by intracellular calcium ([Ca(2+)]i) oscillations.
- In mouse eggs, these oscillations cease, correlating with reduced inositol 1,4,5-trisphosphate receptor (IP3R) responsiveness and IP3R type 1 (IP3R-1) down-regulation.
Purpose of the Study:
- To investigate the specific signaling pathway that induces IP3R-1 down-regulation during mammalian egg fertilization.
- To identify the molecular mechanisms underlying IP3R-1 regulation post-fertilization.
Main Methods:
- Mouse eggs were treated with various Ca(2+)-releasing agonists or injected with substances like sperm fractions (SF) or adenophostin A.
- Western blotting was employed to quantify IP3R-1 immunoreactivity.
- The role of the proteasome pathway was assessed using lactacystin, a proteasome inhibitor.
Main Results:
- Single [Ca(2+)]i rises induced by ethanol or ionomycin did not down-regulate IP3R-1.
- [Ca(2+)]i oscillations triggered by SF or adenophostin A mimicked or exceeded fertilization-induced IP3R-1 down-regulation.
- Thimerosal also initiated IP3R-1 down-regulation, while SrCl(2)-induced oscillations did not.
- Proteasome inhibition with lactacystin blocked IP3R-1 degradation, implicating the proteasome pathway.
Conclusions:
- Persistent stimulation of the phosphoinositide pathway, as occurs during fertilization by sperm or SF injection, leads to IP3R-1 down-regulation in mouse eggs.
- The proteasome pathway is the primary mechanism for IP3R-1 degradation following fertilization-induced signaling.
- Understanding this pathway is crucial for comprehending mammalian egg activation and early development.
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