Cyclic nucleotide phosphodiesterase 3A–deficient mice as a model of female infertility
Silvia Masciarelli, … , Marco Conti, Vincent Manganiello
Silvia Masciarelli, … , Marco Conti, Vincent Manganiello
Published July 15, 2004
Citation Information: J Clin Invest. 2004;114(2):196-205. https://doi.org/10.1172/JCI21804.
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Article Reproductive biology

Cyclic nucleotide phosphodiesterase 3A–deficient mice as a model of female infertility

Abstract

Since cAMP blocks meiotic maturation of mammalian and amphibian oocytes in vitro and cyclic nucleotide phosphodiesterase 3A (PDE3A) is primarily responsible for oocyte cAMP hydrolysis, we generated PDE3A-deficient mice by homologous recombination. The Pde3a–/– females were viable and ovulated a normal number of oocytes but were completely infertile, because ovulated oocytes were arrested at the germinal vesicle stage and, therefore, could not be fertilized. Pde3a–/– oocytes lacked cAMP-specific PDE activity, contained increased cAMP levels, and failed to undergo spontaneous maturation in vitro (up to 48 hours). Meiotic maturation in Pde3a–/– oocytes was restored by inhibiting protein kinase A (PKA) with adenosine-3′,5′-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS) or by injection of protein kinase inhibitor peptide (PKI) or mRNA coding for phosphatase CDC25, which confirms that increased cAMP-PKA signaling is responsible for the meiotic blockade. Pde3a–/– oocytes that underwent germinal vesicle breakdown showed activation of MPF and MAPK, completed the first meiotic division extruding a polar body, and became competent for fertilization by spermatozoa. We believe that these findings provide the first genetic evidence indicating that resumption of meiosis in vivo and in vitro requires PDE3A activity. Pde3a–/– mice represent an in vivo model where meiotic maturation and ovulation are dissociated, which underscores inhibition of oocyte maturation as a potential strategy for contraception.

Authors

Silvia Masciarelli, Kathleen Horner, Chengyu Liu, Sun Hee Park, Mary Hinckley, Steven Hockman, Taku Nedachi, Catherine Jin, Marco Conti, Vincent Manganiello

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Figure 6

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Reinitiation of meiosis in Pde3a–/– ooctyes. (A) Activation of MPF and M...
Reinitiation of meiosis in Pde3a–/– ooctyes. (A) Activation of MPF and MAPK in Pde3a–/– oocytes that had resumed meiosis. Harvested Pde3a+/+ or Pde3a–/– oocytes were incubated in L-15 medium (containing 5% FBS and 100 IU/ml penicillin/streptomycin) with or without 5.0 mM Rp-cAMPS for 2 hours as indicated and then in the absence of Rp-cAMPS for the indicated time prior to assay of MPF and MAPK, as described in Methods. MPF and MAPK were activated in Pde3a–/– oocytes that had been incubated with Rp-cAMPS, but not in untreated Pde3a–/– oocytes. (B) In vitro fertilization of Pde3a–/– oocytes that had resumed meiosis. Pde3a–/– oocytes were incubated with 10 mM Rp-cAMPS for 2 hours, washed, and incubated overnight (20 h) in L-15 medium at 37°C and then with acrosome-reacted sperm for 4 or 24 hours as described in Methods. After in vitro fertilization, a two-cell–stage embryo was generated, indicating that Pde3a–/– oocytes rescued by treatment with the PKA inhibitor were physiologically competent for fertilization and able to develop to at least a two-cell-stage embryo. (C) Reinitiation of meiosis by microinjection of Pde3a–/– oocytes with PKA inhibitor peptide or cdc25b mRNA. Denuded Pde3a–/– oocytes were injected with MES or antisense cdc25b mRNA, as controls, or with PKA inhibitor peptide (PKI) (6 mg/ml) or sense cdc25b mRNA (400 ng/μl). GVBD was assessed after 4 and 24 hours incubation. GVBD was significantly increased in oocytes injected with either PKI or cdc25 mRNA, but not in oocytes injected with vehicles.