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Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing
Published on: October 11, 2015
Generation of mouse oocytes defective in cAMP synthesis and degradation: endogenous cyclic AMP is essential for
Sergio Vaccari1, Kathleen Horner, Lisa M Mehlmann
1Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University, CA 94305, USA.
Abstract:
Although it is established that cAMP accumulation plays a pivotal role in preventing meiotic resumption in mammalian oocytes, the mechanisms controlling cAMP levels in the female gamete have remained elusive. Both production of cAMP via GPCRs/Gs/adenylyl cyclases endogenous to the oocyte as well as diffusion from the somatic compartment through gap junctions have been implicated in maintaining cAMP at levels that preclude maturation. Here we have used a genetic approach to investigate the different biochemical pathways contributing to cAMP accumulation and maturation in mouse oocytes. Because cAMP hydrolysis is greatly decreased and cAMP accumulates above a threshold, oocytes deficient in PDE3A do not resume meiosis in vitro or in vivo, resulting in complete female infertility. In vitro, inactivation of Gs or downregulation of the GPCR GPR3 causes meiotic resumption in the Pde3a null oocytes. Crossing of Pde3a(-/-) mice with Gpr3(-/-) mice causes partial recovery of female fertility. Unlike the complete meiotic block of the Pde3a null mice, oocyte maturation is restored in the double knockout, although it occurs prematurely as described for the Gpr3(-/-) mouse. The increase in cAMP that follows PDE3A ablation is not detected in double mutant oocytes, confirming that GPR3 functions upstream of PDE3A in the regulation of oocyte cAMP. Metabolic coupling between oocytes and granulosa cells was not affected in follicles from the single or double mutant mice, suggesting that diffusion of cAMP is not prevented. Finally, simultaneous ablation of GPR12, an additional receptor expressed in the oocyte, does not modify the Gpr3(-/-) phenotype. Taken together, these findings demonstrate that Gpr3 is epistatic to Pde3a and that fertility as well as meiotic arrest in the PDE3A-deficient oocyte is dependent on the activity of GPR3. These findings also suggest that cAMP diffusion through gap junctions or the activity of additional receptors is not sufficient by itself to maintain the meiotic arrest in the mouse oocyte.
Insights
Mouse oocytes require specific pathways to prevent premature maturation. Genetic studies reveal GPR3 is crucial for maintaining meiotic arrest by regulating cAMP levels, impacting female fertility.
Area of Science:
- Reproductive Biology
- Molecular Endocrinology
- Cellular Signaling
Background:
- Cyclic adenosine monophosphate (cAMP) accumulation is vital for preventing meiotic resumption in mammalian oocytes.
- Mechanisms controlling oocyte cAMP levels, including endogenous production and somatic diffusion, remain incompletely understood.
Purpose of the Study:
- To genetically investigate biochemical pathways regulating cAMP and meiotic maturation in mouse oocytes.
- To elucidate the roles of PDE3A and GPR3 in oocyte meiotic arrest.
Main Methods:
- Utilized a genetic approach involving knockout mouse models (Pde3a, Gpr3, and double mutants).
- Assessed oocyte maturation in vitro and in vivo.
- Measured cAMP levels and evaluated metabolic coupling between oocytes and granulosa cells.
Main Results:
- Oocytes deficient in PDE3A exhibit high cAMP levels and are arrested from meiosis, causing infertility.
- In Pde3a-deficient oocytes, GPR3 inactivation or Gs inactivation restores meiotic resumption.
- Double mutants (Pde3a-/- Gpr3-/-) show partial fertility recovery with premature oocyte maturation, indicating GPR3 acts upstream of PDE3A.
- Metabolic coupling and cAMP diffusion via gap junctions were not impaired.
Conclusions:
- GPR3 is epistatic to PDE3A in regulating oocyte cAMP levels and maintaining meiotic arrest.
- Fertility and meiotic arrest in PDE3A-deficient oocytes are dependent on GPR3 activity.
- cAMP diffusion or other receptors are insufficient alone to maintain meiotic arrest in mouse oocytes.
